Abstracts - International Mammalian Genome Society

Transcription

Aegean Conferences Series-Vol. 54
AEGEAN CONFERENCES
Linking the international scientific community
Bringing the humanity scholars together
together with the
INTERNATIONAL MAMMALIAN
GENOME SOCIETY
24th International Mammalian
Genome Conference
October 17-21, 2010
Aldemar: Knossos Royal Village Conference Center
Hersonissos, Crete, Greece
24th International Mammalian Genome Conference
Aldemar Knossos Royal Village
October 17-21, 2010
Hersonissos, Crete, Greece
COMMITTEES
International Mammalian Genome Society Secretariat
Maja Bucan, Past President (2010)
Karen Steel, President (2010)
David Threadgill, Vice-President (President 2011)
Kent Hunter (2010)
David Beier (2011)
Ian Jackson (2011)
Nancy Jenkins (2011)
Simon Foote (2012)
Xavier Montagutelli (2012)
Darren Logan (2011) VM Chapman award winner
Nomination and Election Committee
Kathryn Hentges (2010)
Bruce Herron (2010)
Amy Moser (2010)
Rosemary Elliott (2011)
Beverly Mock (2011)
Thomas Saunders (2011)
Committee on the Standardized Genetic Nomenclature for Mice
Janan Eppig, Chair
Ruth Arkell
Piero Carninci
Sally Cross
Deanna Church
Jonathan Flint
Simon Foote
Jiri Forejt
Teresa Gunn
David Largespada
Lois Maltais
3
Amy Moser
Jean-Jacques Panthier
David Threadgill
Wolfgang Wurst
Aamir Zuberi
Local Organizing Committee
Joe Nadeau
Director of Research and Academic Affairs
Institute for Systems Biology
1441 North 34th Street
Seattle, WA 98103-8904
Tel: 1-206-732-1207
E-mail: [email protected]
Rudi Balling
Director
Luxembourg Centre for Systems Biomedicine
Universite' du Luxembourg
Campus Limpertsberg
162a Rue de la Faiencerie
L-1511 Luxembourg
David J. Galas
Professor & Senior Vice President,
1441 N. 34th Street,
Seattle, WA 98103
George Kollias
President & Scientific Director
B.S.R.C. "Alexander Fleming"
P.O. Box 74145
16602, Varkiza, Greece
John D. Lambris
Dr. Ralph and Sallie Weaver Professor of Research Medicine
Founder & Executive Director of AegeanConferences
University of Pennsylvania
422 Curie Boulevard
Philadelphia, PA 19104
E-mail:[email protected]
www.lambris.net
4
Official Journal of the
International Mammalian Genome Society
Sponsored by the
International Committee for
Standardized Genetic Nomenclature
Editors
Joseph H. Nadeau
Director of Research and Academic
Affairs
1441 North 34th Street
Seattle, WA 98103-8904
USA
Steve D.M. Brown
Medical Research Council
Mammalian Genetics Unit
Harwell, Didcot
Oxfordshire OX11 ORD
United Kingdom
Informatics Editors
Janan T. Eppig, Bar Harbor, Maine
USA
Reviews Editor
Maja Bucan, Philadelphia, Pennsylvania,
USA
Managing Editor
Louise Tinsley, Oxfordshire, UK
IMGS Business Manager
Darla Miller, Chapel Hill, NC, USA
Editors
D. Adams, Cambridge, United Kingdom
M.D. Adams, Cleveland, Ohio, USA
T. Aitman, London, United Kingdom
L. Andersson, Uppsala, Sweden
R. Arkell, Canberra, Australia
P. Avner, Paris, France
E. Bailey, Lexington, Kentucky, USA
R. Balling, Braunschweig, Germany
D.R. Beier, Boston, Massachusetts, USA
B. Beutler, La Jolla, California, USA
S. Camper, Ann Arbor, Michigan, USA
G. Churchill, Bar Harbor, Maine, USA
N.G. Copeland, Singapore
E. Fisher, London, United Kingdom
S. Foote, Hobart, Tasmania, Australia
W. Frankel, Bar Harbor, Maine, USA
D. Gauguier, Oxford, United Kingdom
M. Georges, Liege, Belgium
C. Goodnow, Canberra, Australia
K.A. Gould, Omaha, Nebraska, USA
E.D. Green, Bethesda, Maryland, USA
P. Gros, Montreal, Quebec, Canada
Y. Herault, Orleans, France
M. Hrabe´ de Angelis, Neuherberg, Germany
D.A. Hume, Edinburgh, UK
I.J. Jackson, Edinburgh, United Kingdom
N. Jenkins, Singapore
M. Justice, Houston, Texas, USA
G. Kelsey, Cambridge, UK
H. Khatib, Madison, Wisconsin, USA
E. Lander, Cambridge, Massachusetts, USA
M. Lyon, Oxfordshire, United Kingdom
T. Magnuson, Chapel Hill, NC, USA
K. Murphy, College Station, Texas, USA
D. Norris, Oxfordshire, United Kingdom
E.A. Ostrander, Bethesda, Maryland, USA
W.J. Pavan, Bethesda, Maryland, USA
D. Pomp, Chapel Hill, North Carolina, USA
L.C. Schalkwyk, London, United Kingdom
T. Shiroishi, Shizuoka-ken, Japan
K. Steel, Cambridge, United Kingdom
C.K. Tuggle, Ames, Iowa, USA
E. Whitelaw, Brisbane, Australia
L. Wicker, Cambridge, United Kingdom
J. Womack, College Station, Texas, USA
J.G. Zhang, Ann Arbor, Michigan, USA
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TABLE OF CONTENTS
General Information
8
Program Outline
10
Program
13
Abstracts
35
Bio Informatics Workshop Program
187
List of Participants
190
Sponsors
195
Upcoming Events
196
Conference Map
197
Hotel Map
198
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AWARDS
The Secretariat, members of The International Mammalian Genome Society,
and Aegean Conferences are proud of the students and postdoctoral fellows who
present oral or poster presentations at the annual meeting. The outstanding
contributions of these young scientists are recognized through several awards.
The first of these awards is the Verne Chapman Young Scientist Award, which
is given to the most outstanding oral presentation from a postdoctoral fellow or
student. This is a monetary award of $500 that reflects Dr. Chapman’s
dedication to mentoring junior scientists. This award also includes a two year
term on the secretariat. Two second place monetary awards of $250
each are given this year by The Genetics Society of America.
A large group of publishing companies sponsor (see page 195) this year’s
presentation prizes, which are a one year subscription to their journal. Genesis is
donating books. These awards are given to the most outstanding poster
presentations by graduate students and postdoctoral fellows, and are chosen by
members of the IMGS Secretariat during the course of the meeting.
7
GENERAL INFORMATION
Accommodations
The conference participants will be staying at the Aldemar: Knossos Royal
Village (A' class), which is located in Hersonissos, 25 km from the airport.
Arrival and Check-in
It is expected that attendees will arrive/check-in on Sunday after 2:00 PM.
Anyone arriving after 10:30 pm on Sunday must make alternative dinner
arrangements.
Conference Venue
The Aldemar: Knossos Royal Village Conference Center is located at the hotel.
(#29 on Hotel Map)
Handicapped Accessibility
The hotel and conference facilities are handicapped accessible.
Posters
Posters should be mounted Monday, October 18, on the designated boards in
Conference Hall II and removed at the closing of the conference. The
dimensions of the boards are 90 cm wide x 120 cm high. Adhesive tape for
mounting the posters on the boards will be available at the poster area. Odd
number posters will be viewed at session 1. Even number posters will be viewed
at session 2. Posters will be on display for the entire duration of the meeting.
(#29 on Hotel Map)
Oral Presentations
Speakers are asked to bring their USB flash drive or CD to the conference
reception desk at least 30 minutes prior to the session. The allocated time for all
presentations, unless otherwise indicated, includes five minutes Q&A.
Tour to Knossos and Museum
Buses for the city & archaeological tour will be leaving from the hotel on
Tuesday, October 19 at 2:00p.m. Participants will be picked up at the hotel front
lobby at that time. Badges are required for participation.
Breakfast
Breakfast for registered participants and registered accompanying persons will be
served at the Aldemar Knossos Royal Village hotel main restaurant during the
hours scheduled in this program. (#2 on Hotel Map) 8
Lunches/Informal Discussions
Lunches for registered participants and registered accompanying persons will be
served during the times indicated in this program at the Main Restaurant.
Badges are required for admission. (#2 on Hotel Map)
Dinners/Informal Discussions
Three of the dinners/informal discussion will be served during the times
indicated in the program. Vegetarian entrees are available only upon request in
advance of the meeting. Please contact [email protected].
Internet, Phone, & Computer
Wireless Internet access is located throughout the conference center free of
charge during meeting hours. There are computers available in the hotel’s
business center for the use of the attendees. Phone service is available for an
additional charge from your hotel room.
Other Information
Animals are not permitted on site.
Badges are required for admission to all events.
All Aegean Conferences events are non-smoking.
No Photographs and Recording Devices
Participants are not allowed to photograph and/or record using cameras, mobile
telephones and other recording devices during the slide and poster presentations.
Tour and Travel Information
A desk operated by the personnel of our official travel agency
Conferex LLC, P.O. Box 12, Wynnewood, PA 19096, USA
Tel.: 1-484-685-0558, FAX: 1-610-771-4224
E-mail: [email protected], www.conferex.com
ERA Ltd., 17, Asklipiou Str., 106 80 Athens-Greece
Tel.: 30-210-363-4944, FAX: 30-210-3631690, E-Mail: [email protected]
will be located at the conference center throughout the meeting.
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PROGRAM OUTLINE
2:00 PM – 6:00 PM
7:00 AM-8:00 AM
8:00 AM-9:00 AM
9:00 AM-12:30 PM
Saturday, October 16, 2010
Secretariat Meeting
Theater Room Hotel Lobby
Sunday, October 17, 2010
Breakfast
Registration: Bioinformatics Workshop
Lobby I
Bioinformatics Workshop (see appendix 1)
Tutorial 1 Room Orpheas
Tutorial 2 Room Apollon
Tutorial 3 Room Hermes
12:30 PM-1:00 PM
Coffee Break
11:00 AM
Registration: Student Satellite Symposium
Lobby I
Student Satellite Symposium Session 1
Conference Hall I
1:00 PM-2:45 PM
2:45 PM-3:30 PM
Coffee Break
3:30 PM-5:30 PM
Conference Hall I
4:00 PM – 7:00 PM
24th IMGS Registration Open
Lobby I
7:00 PM-10:30 PM
Welcome Reception and Dinner
7:00 AM-8:20 AM
Monday, October 18, 2010
Breakfast
8:00 AM-5:30 PM
Lobby I
8:20 AM-8:30 AM
Official 24th IMGS Opening
10
8:30 AM-10:30 AM
Session 1: Infection and Immunity
Conference Hall I
10:30 AM–12:30 PM Coffee Break/ Exhibition and Poster Session 1
Odd Numbered Posters
Conference Hall II
12:00 PM-1:30 PM
Editorial Board Meeting with lunch
12:30-1:30 PM
Lunch
1:30 PM-3:30 PM
Session 2: Modeling Disease-Development,
Metabolism and Physiology
Conference Hall I
3:30 PM-4:00 PM
Coffee Break
4:00 PM-5:00 PM
IMGS Business meeting (all welcome)
Conference Hall I
5:00 PM-6:00 PM
Verne Chapman Lecture
Conference Hall I
6:30 PM-9:30 PM
Dinner
7:00 AM-8:30 AM
Tuesday, October 19, 2010
Breakfast and Mentor Breakfast
8:30 AM-10:00 AM
Session 3: Neuroscience, Behavior, and Sensory
Systems I
Conference Hall I
10:00 AM
Session 4: Large Scale and Genome-wide Resources
Conference Hall I
10:30AM
Coffee Break
11:00 AM
Session 4 continued: Large Scale and Genome-wide
Resources
1:00 PM-2:00 PM
Lunch
11
2:00 PM-5:00 PM
Guided Tour of the Knossos Archeological Site
Buses depart hotel lobby at 2:00 PM
5:00 PM-6:30 PM
SPECIAL SESSION
Human Disease and Mouse Models
Conference Hall I
6:30 PM-9:30 PM
Dinner
7:00 AM-8:30 AM
Wednesday, October 20, 2010
Breakfast
8:30 AM-10:30AM
Session 5:
Epigenetics, Neoplasia and Aging I
Sponsored by The Ellison Medical
Foundation
Conference Hall I
10:30 AM-12:30 PM Coffee Break/Exhibition and Poster Session 2
Even Numbered Posters
Conference Hall II
12:30 PM-1:30 PM
12:00 PM-1:30 PM
Lunch
Secretariat meeting with lunch
1:45 PM-3:15 PM
Session 6:
Epigenetics, Neoplasia and Aging II
Foundation
Conference Hall I
3:15 PM-3:45 PM
Coffee Break
3:45 PM-4:30 PM
Session 7: Neuroscience, Behavior and Sensory
Systems II
Conference Hall I
7:00 PM
Farewell Dinner
12
PROGRAM
13
PROGRAM
2:00 PM – 6:00 PM
7:00 AM-9:00 AM
Saturday, October 16, 2010
Secretariat Meeting
Sunday, October 17, 2010
Breakfast
8:00 AM-9:00 AM
Registration: Bioinformatics Workshop
Lobby I
9:00 AM-12:30 PM Bioinformatics Workshop (see appendix 1)
Tutorial 1 Room Orpheas
Tutorial 2 Room Apollon
Tutorial 3 Room Hermes
12:30 PM-1:00 PM
Coffee Break
11:00 AM
Registration: Student Satellite Symposium
Lobby 1
Chairs: Karen Steel, Maja Bucan
Conference Hall I
1:00 PM
1
1:15 PM
2
1:30 PM
3
1:45 PM
4
2:00 PM
5
The F2 population for dissecting host susceptibility to
periodontitis
Ariel Shusterman, Yael Houri-Haddad, Ervin I Weiss,
and Fuad A Iraqi
Mouse genetic model of cardiovirulent coxsackievirus
B3 infection
Sean A. Wiltshire and Silvia M. Vidal
Association between spatial proximity and functional
similarity in human genome
Ekaterina Khraneeva, Andrey Mironov, and Mikhail
Gelfand
Olfactory receptor gene family evolution in marsupials
Amir Mohammadi, Margaret L. Delbridge, and Jennifer
A. Marshal Graves
Advancing paternal age is associated with deficits in
social and exploratory behaviours in the offspring
Rebecca G. Smith, Rachel L. Kember, Leonard C.
15
2:15 PM
6
2:30 PM
7
2:45 PM
Schalkwyk, Joseph D Buxbaum, Abraham Reichenberg,
Cathy Fernandes, and Jonathan Mill
Genome-wide differential methylation patterns in
intersubspecific hybrid mice
John D. Calaway, Hyuna Yang, Elena de la CasaEsperon, Megan E. Hanson, Ezequiel C. Cambranis,
Maria A. Perez-Lamigueiro, David L. Aylor, Leonard
McMillan, Gary A. Churchill, and Fernando PardoManuel de Villena
Optimization of a genome-wide, allele-specific DNA
methylation assay for the mouse
John P. Didion, John D. Calaway, Isa Kemal Pakatci,
Leonard McMillan, and Fernando Pardo-Manuel de
Villena
Coffee Break
Chairs: David Beier, David Threadgill
Conference Hall I
3:30 PM
8
3:45 PM
9
4:00 PM
10
4:15 PM
11
4:30 PM
12
Characterisation
of
laterality
and
cardiac
developmental defects in the K27 mutant mouse
Louise Stephen, Karen Mitchell, and Kathryn Hentges
Kyoto Epileptic Rat (KER): A novel rat model of Kcna1
mutation showing spontaneous seizures and muscle
involuntry contractions
Saeko Ishida, Tomoji Mashimo, Takeshi Nishio, Yu
Sakamoto, Shuji Kaneko, and Tadao Serikawa
Identifying modifier genes of mpnsts in the Nf1;p53cis
mouse model of neurofibromatosis type 1
Jessica A Van Schaick, Keiko Akagi, Sandra Burkett,
Christina DiFabio, Robert Tuskan, Jessica Walrath, and
Karlyne Reilly
A novel genetic model of osteoporosis by
overexpression of human RANKL in transgenic mice
Alexandra Niti, R Dacquin, V Rinotas, P Jurdic, and E
Douni
Positive selection might contribute to functional
divergence of NAIPs following gene duplication in
mammals during the evolution
Zhaoliang Su, Chenglin Zhou, Jianguo Chen, Shengjun
Wang, and Huaxi Xu
16
4:45 PM
13
5:00 PM
14
5:15 PM
15
A forward genetics approach identifies a novel member
of the Dnajc family that causes neuromuscular disease
in mice
Fotios Ioakeimidis, Vagelis Rinotas, Eleni Makrinou,
George Kollias, and Eleni Douni
Gene expression studies of ncRNAs within Odz4 locus
Tyler Buit, Chiao-Ling Lo, Feichen Shen, and Amy C.
Lossie
Early life stress is associated with altered behavior and
epigenetic alterations to Nr3c1, Avp and Nr4a1
Rachel L. Kember, Emma Dempster, Leonard C.
Schalkwyk, Jonathan Mill and Cathy Fernandes
4:00 PM -7:00 PM
Lobby I
7:00 PM-10:30 PM
Welcome Reception and Dinner
7:00 AM-8:20 AM
Monday, October 18, 2010
Breakfast
8:00 AM-5:30 PM
Lobby I
8:20 AM
Official 24th IMGS Opening
Session 1: Infection and Immunity
Chairs: Simon Foote, Karen Svenson
Conference Hall I
8:30 AM
16 Stress mechanisms, inflammation, infection and host
resistance
Katia P. Karalis
9:15 AM
17 Physiologic consequences of constitutive MTOR
inhibition: alterations in cell/organ size and immune
cell development
Shuling Zhang, Julie A. Readinger, Wendy DuBois,
Mirkka Janka-Junttila, Richard Robinson, Magaret
Pruitt, Val Bliskovsky, Julie Z. Wu, Kaori Sakakibara,
Jyoti Patel, Carole A. Parent, Lino Tessarollo, Pamela L.
Schwartzberg, and Beverly A. Mock
9:30 AM
18 Severe Combined Immunodeficiency (SCID) rats
generated by zinc finger nuclease technology
Tomoji Mashimo, Akiko Takizawa, Birger Voigt,
17
9:45 AM
19
10:00 AM
10:15 AM
Kazuto Yoshimi, Hiroshi Hiai, Takashi Kuramoto, and
Tadao Serikawa
A recombination hotspot leads to sequence variability
within a novel gene and contributes to complex disease
susceptibility
Iris KL Tan, Leanne Mackin, Nancy Wang, Anthony T
Papenfuss, Colleen M Elso, Michelle P Ashton, Belinda
Phipson, Melanie Bahlo, Terrence P Speed, Gordon K
Smyth, Grant Morahan, and Thomas C Brodnicki
Selected Student Talk
10:30 AM
Coffee Break/ Exhibition and Poster Session 1
Odd Numbered Posters
Conference Hall II
12:30 PM-1:30 PM
Lunch
12:00PM-1:30 PM
Mammalian Genome Editorial Board Meeting with
Lunch
Session 2: Modeling Disease: Development, Metabolism and Physiology
Chairs: Nancy Jenkins, Gail Herman
Conference Hall I
1:30 PM
20
1:45 PM
21
2:00 PM
22
2:15 PM
23
A sensitized suppressor screen to identify modifier
genes for factor V leiden-dependent thrombosis in the
mouse
Randal Westrick, Goujing Zhu, Sara Manning, Angela
Yang, David Siemieniak, and David Ginsburg
Genetic and dietary controls of alternative exon use
and mRNA levels in the systems architecture of dietinduced metabolic diseases
Sabrina H. Spiezio, Annie Hill-Baskin, Karen Fitch,
Keith Jones, Michael H. Shapero, and Joseph H. Nadeau
A gene driven ENU mutagenesis screen reveals a novel
method of ZIC protein nuclear localization
R Ali, H Bellchambers, N Warr, D Quwailid, P Denny
and Ruth Arkell
Non-muscle myosin IIB is required for formation of
the coronary vasculature
Karen Mitchell, C Clowes, Louise Stephen, and Kathryn
18
2:30 PM
24
2:45 PM
25
3:00 PM
26
3:15 PM
27
E Hentges
Short circuit, a homozygous lethal ENU mutation that
affects lung development
Michael J. Parsons, Laura Yates, Jessica Edwards,
Lauren Chessum, Charlotte H. Dean, and Patrick M.
Nolan
Identification of the Danforth’s short tail mutation
using next generation sequencing
Christopher N. Vlangos, Amanda N. Siuniak, Dan
Robinson, Arul M. Chinnaiyan, James Cavalcoli, Robert
H. Lyons, and Catherine E. Keegan
A novel role for Atmin, controlling ciliogenesis
through modulation of dynein light chain expression
Paraskevi Goggolidou, Jonathan Stevens, Gabrielle
Wheway, Antonella Di Paolo, Rosario Romero, James
Briscoe, Colin Johnson, and Dominic Norris
Genetic analysis of complex traits in the emerging
collaborative cross
David L. Aylor, William Valdar,, Wendy FouldsMathes, Ryan J. Buus, Ricardo A. Verdugo,, Ralph S.
Baric, Martin T. Ferris, Jeffrey A. Frelinger, Mark
Heise, Matt B. Frieman, Lisa E. Gralinski, Timothy A.
Bell, John P. Didion, Kunjie Hua, Derrick L.
Nehrenberg, Christine L. Powell, Jill Steigerwalt,
Yuying Xie, Samir N.P. Kelada, Francis S. Collins,
Ivana V. Yang, David A. Schwartz, Lisa A. Branstetter,
Elissa J. Chesler, Darla R. Miller, Jason Spence, Eric Yi
Liu, Leonard McMillan, Abhishek Sarkar, Jeremy
Wang, Wei Wang, Qi Zhang, Karl W. Broman, Ron
Korstanje, Caroline Durrant, Richard Mott, Fuad A.
Iraqi, Daniel Pomp, David Threadgill, Fernando PardoManuel de Villena, and Gary A. Churchill
3:30 PM-4:00 PM
Coffee Break
4:00 PM-5:00 PM
IMGS Business meeting (all welcome)
Conference Hall I
5:00 PM –
6:00 PM
Verne Chapman Lecture
Realising the potential of mouse disease models – from
pathway to therapy
Steve D.M. Brown
Conference Hall I
28
19
6:30 PM-9:30 PM
Dinner
7:00 AM-8:30 AM
Tuesday, October 19, 2010
Breakfast and Mentor Breakfast
Session 3: Neuroscience, Behavior, and Sensory Systems I
Chairs: Ian Jackson, Elizabeth Bryda
Conference Hall I
8:30 AM
29
8:45 AM
30
9:00 AM
31
9:15 AM
32
9:30 AM
33
9:45 AM
Importance of Cadm1 and cell adhesion in depressive
behavior
C Santos, B Miller, M Pletcher, A Su, L Tarantino, and
Tim Wiltshire
An ENU sensitization screen to determine the
physiological function of α - synuclein
Deborah E Cabin, M Casey, and D Zou
Genomic resources in Peromyscus – mapping the
audiogenic seizure sensitivity locus
Gabor Szalai, Janet Crossland, Jay Coleman, and
Michael Felder
Mutations in the PDZ domain containing protein
Gipc3 cause progressive sensorineural degeneration
(Ahl5 and jams1) in mice and recessive hearing
impairment in humans (DFNB95)
Nikoletta Charizopoulou, Andrea Lelli, Margit
Schraders, Kausik Ray, Ronald J.C. Admiraal, Harold R.
Neely, Joseph R. Latoche, John K. Northup, Hannie
Kremer, Jeffrey R. Holt, and Konrad Noben-Trauth
A mutation in the gene encoding mitochondrial Mg2+
channel MRS2 results in demyelination in rats
Takashi Kuramoto, Mitsuru Kuwamura, Satoko Tokuda,
Takeshi Izawa, Yoshifumi Nakane, Kazuhiro Kitada,
Masaharu Akao, Jean-Louis Guénet, and Tadao
Serikawa
Session 4: Large Scale and Genome-wide Resources
Chairs: David Beier, Joe Nadeau
Conference Hall I
10:00 AM
34
10:15 AM
35
Annotation of long non-coding RNA transcripts
Mark Thomas and Jennifer Harrow
The JAX Cre Repository: Improving the utility of Cre
20
driver strains
Stephen A. Murray, Caleb Heffner, Michael Sasner,
Cathleen Lutz, Brandon Grossman, Stephen Rockwood,
Yashoda Sharma, and Leah Rae Donahue
10:30 AM
Coffee Break
11:00 AM
36
11:15 AM
37
11:30 AM
38
11:45 AM
39
12:00 Noon
40
12:15 PM
41
12:30 PM
42
12:45 PM
43
Mouse genomes project: map of SNPS and short indels
Petr Danecek, Gan Xiangchao, Thomas M Keane, James
Stalker, Binnaz Yalcin, Martin Goodson, Sendu
Balasubramaniam, Kim Wong, Guy Slater, Andreas
Heger, Eleazar Eskin, Nick Furlotte, Chris Ponting,
Jonathan Flint, and David J Adams
Use of PiggyBac-mediated transient transgenic RNAi
expression for rapid characterization of gene function
during embryonic development
David R. Beier, Yuko Fujiwara, Shannon W. Davis,
Haiyan Qiu, Thomas L. Saunders, Stuart Orkin, Sally A.
Camper, and Bryan C. Bjork
The Sanger Institute Mouse Genetics Programme
Ramiro Ramirez-Solis, J White, E Ryder, R Houghton,
and J Bottomley
Next level systemic phenotyping of mice
Martin Hrabé de Angelis and the German Mouse Clinic
consortium
Portal to large-scale mouse knockout resources
William Skarnes
Tooling up MGI for a deluge in mouse phenotype and
disease model data
Anna Anagnostopoulos, Janan Eppig, and Mouse
Genome Informatics Group
New approaches to conditionality
Aris N. Economides, David Frendewey, Peter Yang,
David M. Valenzuela, Andrew J. Murphy, and George
D. Yancopoulos
Mouse metabolic phenotyping centers: National
consortium focusing on diabetic complications
Renee LeBoeuf
1:00 PM-2:00 PM
Lunch
2:00 PM-5:00 PM
Guided Tour of the Knossos Archeological Site
Buses depart hotel lobby at 2:00 PM
21
5:00 PM-6:30 PM
5:00 PM
44
5:45 PM
45
SPECIAL SESSION
Human Disease and Mouse Models
Conference Hall I
Lupus
Edward Wakeland
Cancerous microRNAs and regulatory RNA binding
proteins
Reuven Agami
6:30 PM
Dinner
7:00 AM-8:30 AM
Wednesday, October 20, 2010
Breakfast
Session 5: Epigenetics, Neoplasia and Aging I
Sponsored by The Ellison Medical Foundation
Chairs: Teresa Gunn, Kent Hunter
Conference Hall I
8:30 AM
46 Genomic imprinting: Insights from the Gnas cluster
Jo Peters
9:15 AM
47 The battle of the signals: Non-coding RNAs vs.
enhancers in the transcriptional regulation of the
Kcnq1 imprinted region
Anjali Raval, Lisa Korostowski, and Nora Engel
9:30 AM
48 An identity crisis: Abnormal male germ cell
development and the initiation of testicular cancer
Jason Heaney, Jean Kawasoe, Megan Michelson, and
Joseph Nadeau
9:45 AM
49 Age-associated change of energy metabolism are
genetically dissected by mouse inter-subspecific
consomic strains
Toyoyuki Takada, Akihiko Mita, Shigeharu Wakana,
Kazuo Moriwaki, Hiromichi Yonekawa, and Toshihiko
Shiroishi
10:00 AM
50 Genetic regulation of aging related phenotypes and
lifespan in mice
Rong Yuan, S W Tsaih, Q Meng, K Flurkey, J Nautiya,
SB Petkova, MA Bogue, KD Mills, L L Peters, CJ Bult,
CJ Rosen, JP Sundberg, M Parker, DE Harrison, GA
Churchill, and B Paigen
10:15 AM
51 Subspecific origin and haplotype diversity in the
laboratory mouse
Hyuna Yang, Jeremy R Wang, John P Didion, Ryan J
22
Buus, Timothy A Bell, Catherine E Welsh2, François
Bonhomme, Alex Hon-Tsen Yu, Michael W Nachman,
Jaroslav Pialek, Priscilla Tucker, Pierre Boursot,
Leonard McMillan, Gary A Churchill, and Fernando
Pardo-Manuel de Villena
10:30 AM
Coffee Break/Exhibition and Poster Session 2
Even Numbered Posters
Conference Hall II
12:30 PM-1:30 PM
Lunch
12:00 PM-1:30 PM
Secretariat meeting with lunch
Session 6: Epigenetics, Neoplasia and Aging II
Chairs: Beverley Mock, Xavier Montagutelli
Conference Hall I
Foundation
1:45 PM
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2:30 PM
53
2:45 PM
54
3:00 PM
55
3:15 PM
TBA
David Galas
Genetic architecture of hybrid sterility: Matching the
puzzle pieces together
Jiri Forejt, Maria Dzur-Gejdosova, Vaclav John,
Tanmoy Bhattacharyya, Petr Simecek, Sona Gregorova,
Ondrej Mihola, Petr Flachs, and Zdenek Trachtulec
Molecular interactions of dead end (Dnd1)
Angabin Matin, Zhu Rui, KangLi Luo, Chitralekha
Bhattacharya, Michelina Iacovino, Elisabeth Mahen,
Michael Kyba, and Sita Aggarwal
Genetic dissection of metatasis susceptibility converges
on a common mechanism
J Alsarraj, S Winter, N Goldberger, K Mattaini, M
William, L Lukes, R Walker, and Kent W Hunter
Coffee Break
Session 7: Neuroscience, Behaviour and Sensory Systems II
Conference Hall I
3:45 PM
56 Icst is a dominant negative mutation of Lmx1b
Sally H. Cross, Lisa Mckie, Margaret Keighren, Dan
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4:00 PM
57
4:15 PM
58
7:00 PM
Macalinao, Alison L. Kearney, Simon W. John, and Ian
J. Jackson
Elucidating the role of Nsdhl and cholesterol
metabolism in CNS development using a conditional
knockout allele
Gail E Herman, N Bir, L Binkley, K McLarren, C
Boerkoel, and D Cunningham
Genetic, stem cell, and systems analyses of
neurodegenerative diseases
George A Carlson, R Bennett, ME Orr, I Lee, H Yoo, JH Cho, D Hwang, and LE Hood
Farewell Dinner
24
Posters
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67
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69
Stage and cell subtype-specific epigenetic regulation of mammary
gland development
Deanna Acosta, Melissa J Fazzari, John M Greally, and Cristina
Montagna
Toward an unified measure of intraspecific selective pressure
Roberto Amato, Gennaro Miele, Michele Pinelli, and Sergio Cocozza
A new targeted mutation, Caspa, in the Gnas complex shows
hyperactivity and ataxia
Simon Ball, Sally Eaton, Christine Williamson, Charlotte Tibbit, and
Jo Peters
Rat resource and research center
Beth A. Bauer, EC Bryda, CL Franklin, LK Riley, and JK Critser
Loci on chromosome 2 are associated with naïve airway
hyperresponsiveness in CD-1 outbred mice
David R Beier, Andrew Kirby, Mary Prysak, Mark Daly, and Emily
Cozzi
High-resolution map and candidate gene analysis for ébouriffé
(ebo), a hair mutation on mouse Chromosome 2 associated with
infertility
Fernando Benavides, J Jaubert, CJ Perez, J-L Guénet, J Barrera, B
Cole, and CJ Conti
Molecular genetic analysis of the NADPH oxidase of the vestibular
system
Catrina A Spruce, John P Flaherty, Heather E Fairfield and David E
Bergstrom
Mouse models of Costello syndrome
Heather E Fairfield, John P Flaherty, Catrina A Spruce, Leah Rae
Donahue and David E Bergstrom
The application of high-throughput sequencing technologies for
mutation discovery in the Jackson Laboratory’s mouse mutant
resource
Laura G Reinholdt, David E Bergstrom, Muriel T Davisson-Fahey,
Cathleen M Lutz, Michael Sasner, Steven A Murray, Jeff Lake, Steven
Rockwood, Leah Rae Donahue and the MMR team
The role of CAML in the inner ear
Elizabeth C Bryda, Nathan T Johnson, Cynthia L Besch-Williford,
Kevin K Ohlemiller, and Richard J Bram
Interesting phenotypes found as part of the infection challenge in the
Wellcome Trust Sanger Institute’s Mouse Genetics Programme
Simon Clare, Leanne Kane, Lynda Mottram, Jacqui White, Ramiro
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Ramirez – Solis, and Gordon Dougan
The expression of SEPTIN 9 in human breast cancer
Diana Connolly, Zhixia Yang, Elias T. Spiliotis, Maria Castaldi,
Nichelle Simmons, Pascal Verdier-Pinard, and Cristina Montagna
Genetic contribution to liver fibrosis
David DeSantis, Michelle Pritchard, Laura Nagy, Joseph Nadeau, and
Colleen M Croniger
Diet, obesity, and susceptibility to colon polyps
Stephanie K. Doerner and Joseph H. Nadeau
Genetic regulation of fracture risk in inbred mice
Leah Rae Donahue GA Churchill, S-W Tsaih, and WG Beamer
Imprinted XLAS: A new player in bone and adipocyte metabolism
Sally Eaton, Simon Ball, Colin Beechey, Christine Williamson, and Jo
Peters
The Sanger Mouse Genetics Programme: High throughput recessive
lethality screen
Jeanne Estabel, Elizabeth Tuck, Damian Carragher, Jennifer Salisbury,
and Jacqueline K. White on behalf of the Mouse Genetics Programme
Multiple forebrain cis-regulatory elements at the Dlx1/2 locus:
redundant function or complex control mechanisms?
Marc Ekker, Luc Poitras, Man Yu, Noel Ghanem, and Lisa Tran
Novel mutation discovery on ENU mouse mutagenesis by nextgeneration sequencer
Ryutaro Fukumura, Hayato Kotaki, Yuichi Ishitsuka, Takuya Murata,
Shigeru Makino, Yuji Nakai, and Yoichi Gondo
The Sanger Mouse Genetics Programme: High throughput
characterisation of knockout mice for genes involved in glucose
metabolism
Anna-Karin Gerdin, L Roberson, Jacqueline K White on behalf of the
Mouse Genetics Programme
EuroPhenome: Large dataset visualisation, statistical analysis and
data exploratory tool for mouse phenotyping data
Ahmad Hassan, Hugh Morgan, Andrew Blake, Simon Greenaway, The
EUMODIC Consortium, John M. Hancock, and Ann-Marie Mallon
The intragene resource centre, a national centre for mouse genetics,
distribution, archiving and functional imaging
Cecile Fremond, Stephanie Lerondel, Christelle Martin, and Yann
Herault The Down syndrome as a complex trait resulting from the interaction
of dosage sensitive genes unravelled in the mouse model
V Brault, A Duchon, E Dalloneau, P Lopes, I Abizanda, S Luiller, JC
Bizot, C Borel, M Raveau, D Marechal, J Lignon, S Pothion, F
Trovero, V Tybulewicz, EM Fisher, S Antonarakis, M Dierssen, and
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Yann Herault
The European Mouse Mutant Archive (EMMA)
Michael Hagn, G Tocchini-Valentini, Y Hérault, S Brown, U Lendahl,
J Demengeot, M Hrabé de Angelis (EMMA Director), E Birney, R
Ramirez-Solis, JL Mandel, and L Montoliu
Broad based phenotyping approaches for the characterisation of
mutant mouse lines in EUMODIC and the Harwell aging screen
Heena Vanmalibhai Lad, KA Lee, A Blake, H Morgan, A Hassan, S
Greenaway, AM Mallon, HJ Gates, SDM Brown, and The EUMODIC
Consortium
FBXO11 regulates TGF beta signaling through TRP53
Hilda Tateossian, Susan Morse, and Steve DM Brown
The development of the integrated database of mammals in RIKEN
Hiroshi Masuya, Yuko Makita, Koro Nishikata, Yuko Yoshida, Terue
Takatsuki, Kasunori Waki, Nobuhiko Tanaka, Yoshiki Mochizuki,
Norio Kobayashi, Riichiro Mizoguchi, Teiichi Furuichi, Hideya
Kawaji, Daub Carsten, Yoshihide Hayashizaki, Shigeharu Wakana,
Atsushi Yoshiki, Kaoru Fukami-Kobayashi, and Tetsuro Toyoda
Combining clinical and expression QTLs in the context of infection:
new insights into susceptibility to influenza
Gregory A. Boivin, Julien Pothlichet, Emil Skamene, Earl G. Brown,
Robert Sladek, and Silvia M. Vidal
A nature of the blastomer DNA methylation determination
Andrey A. Ivanov
Not all wild-derived Mus spretus mice do resist virulent Yersinia
pestis
Jean Jaubert, Charlotte Leblanc, Charlène Blanchet, Garcia Sylvie,
Elisabeth Carniel, Christian Demeure, Jean-Jacques Panthier, and
Xavier Montagutelli
Evidence of hypothalamic degeneration in the anorectic anx/anx
mouse
IAK Nilsson, S Thams, C Lindfors, A Bergstrand, S Cullheim, T
Hökfelt, and Jeanette E Johansen
Identification of a novel Slc25 member that causes autosomal
recessive ataxia in ENU-mutagenized mice
A Karakostas, F Ioakeimidis, V Rinotas, G Kollias, and Eleni Douni
Consomic analysis of genetic factors related to temporal difference
of home-cage activity between B6 and MSM
Ayako Ishii, Akinori Nishi, Toshihiko Shiroishi, Aki Takahashi, and
Tsuyoshi Koide
Genetic determinants for intramuscular fat content and water
holding capacity in mice selected for high muscle mass
Stefan Kärst and Gudrun A. Brockman
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Discovering novel regulatory pathways of imflammation by ENU
mutagenesis
Philip L Kong, Emma Timms, Andrew Coulson, Kristina BlagoevaHubbard, Paul Potter, and Marc Feldmann
Epigenetic changes in Friedreich ataxia pathogenesis and therapy
S Al-Mahdawi, C Sandi, R Mouro-Pinto, V Ezzatizadeh, and Mark A
Pook
A mouse mutant exhibiting a unique pattern of cochlear inner hair
cell degeneration
Morag A. Lewis, Tracy J. Bussoli, Amy Taylor, Michael A. Cheong,
Cordelia F. Langford, and Karen P. Steel
A comprehensive whole-genome map of endogenous retroviral
elements and their functional effects across 17 laboratory mouse
strains
Thomas M Keane, K Wong, J Flint, W Frankel, David J Adams
Phenotyping of knockout mice using bacterial pathogens as part of
the Wellcome Trust Sanger Institute’s Mouse Genetics Programme
Leanne Kane, Lynda Mottram, Simon Clare, Jacqui White, Ramiro
Ramirez – Solis, and Gordon Dougan
Imprinted methylation vs. imprinted expression – are they
necessarily associated?
John D. Calaway, Hyuna Yang, Megan E. Hanson, Ezequiel C.
Cambranis, Maria A. Perez-Lamigueiro, David L. Aylor, Leonard
McMillan, Gary A. Churchill, Fernando Pardo-Manuel de Villena, and
Elena de la Casa-Esperon
Dual activity on a single locus: Existence of a novel RNA control
mechanism for microRNA-650 and immunoglobulin lambda light
chain variable genes in primates
Sabyasachi Das, Jianxu Li, Masayuki Hirano, Christopher L. Haga,
Murali Gururajan, Götz R.A. Ehrhardt, and Max D. Cooper
Identification of positional candidates for Chr15 obesity quantitative
trait loci using microarray and RT-PCR analysis of F2 congenic
crosses in mice
K Cirnski, M Stanonik, R Keber, P Juvan, R Košir, D Rozman, and
Simon Horvat
Crezoo - the european virtual repository for cre driver strains
Christina Chandras, Michael Zouberakis, Damian Smedley, Nadia
Rosenthal, and Vassilis Aidinis
Host immune responses and genetic factors modulating resistance to
Salmonella enterica serovar typhimurium in the inbred mouse strain
SPRET/Ei: a role for neutrophils
Lien Dejager, Iris Pinheiro, Pieter Bogaert, Liesbeth Huys, and Claude
Libert
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LPS-resistance of the mouse strain SPRET/Ei: an important role of
the X-chromosome
Iris Pinheiro, Lien Dejager, Tina Mahieu, Marnik Vuylsteke, and
Claude Libert
Complete knockout of the adrenocortical dysplasia gene encoding
the shelterin protein TPP1 is associated with telomere dysfunction
and early embryonic lethality
Gail A Osawa, C Harris, T Kibe, T de Lange, S Kalantry, and CE
Keegan
The FaceBase Cre driver project: creating new mouse tool strains for
clefting research
Stephen A. Murray, Jocelyn Sharp, John Flaherty, Thomas Gridley,
and Leah Rae Donahue
Epigenome sequencing comes of age in development, differentiation
and immune regulation mechanisms of mammalian research
Ning Li and Jun Wang
Generation of customized and read-to-use genetically engineered
mice at the institute Clinique de la souris – ICS (The French Mouse
Clinical Institute)
Marie-Christine Birling, Andrée Dierich, Karim Essabri, Lydie
Venteo, Guillaume Pavlovic, Tania Sorg, and Yann Hérault
Pudding – a novel model of glomerularnephritis
Paul K Potter, L Wisby, FW Tam, S Wells, T Hough, HT Cook, M
Cheeseman, and SDM Brown
The Harwell ageing screen
Paul K Potter
Gelsolin plays a role in the actin polymerization complex of hair cell
stereocilia
Philomena Mburu, MR Romero, Helen Hilton, Andrew Parker, Stuart
Townsend, Yoshiaki Kikkawa, and SDM Brown
New sterile model mouse from RIKEN ENU-based gene-driven
mutagenesis
Takuya Murata, Shigeru Makino, Ryutaro Fukumura, and Yoichi
Gondo
Genetic diversity among C57BL/6 substrains based on SNPs
Kazuyuki Mekada, Ikuo Miura, Takehide Murata, Atsushi Toyoda,
Mayu Hirose, Shigeharu Wakana, Kuniya Abe, and Atsushi Yoshiki
Novel ENU screens for immune-related phenotypes
Vera M Ripoll, Marie Hutchison, Paul Potter, and Roger Cox
Grb10 modifies MPNST tumorigenesis in a mouse model of NF1
Karlyne M. Reilly, Jessica Van Schaick, Keiko Akagi, Christina
DiFabio, Robert Tuskan, Sandra Burkett, and Jessica Walrath
Homology mapping: development of the mouse hindlimb
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musculoskeletal model using the human musculoskeletal model and
Scleraxis (Scx)-GFP mouse
Satoshi Oota, Nobunori Kakusho, Yosuke Ikegami, Kazuyuki Mekada,
Koh Ayusawa, Hirotaka Imagawa, Yuichi Obata, Ryutaro Himeno,
Hideo Yokota, Yoshihiko Nakamura, and Atsushi Yoshiki
Mutation identification in mice by exome sequencing
Laura Reinholdt, David Bergstrom, Steve Murray, Michelle Curtain,
Carol Bult, Joel Richardson, Lucy Rowe, Mary Barter, Daniel J.
Gerhardt, Mark D'Ascenzo, Todd Richmond, Tom J. Albert, Jeffrey A.
Jeddeloh, Benjamin Kile, Ivo Gut, Jay Shendure, and Leah Rae
Donahue
Status of the US lines of the Collaborative Cross: Completed lines
Darla R Miller, Ryan J Buus, Jennifer N Shockley, Stephanie D
Hansen, Ginger D Shaw, Terry J Gooch, Jason S Spence, Timothy A
Bell, Catherine E Welsh, Leonard McMillan, Wei Wang, Jeremy
Wang, Eric Yi Liu, Kenneth F Manly, Gary A Churchill, David W
Threadgill, and Fernando Pardo-Manuel de Villena
Whole genome resequencing of Estonian dairy cow (Estonian
Holstein)
Rutt Lilleoja, Ü Jaakma, and S Kõks
Hypothalamic mitochondrial dysfunction, a possible cause of
anorexia
Charlotte Lindfors, Ida A.K. Nilsson, Pablo M. Garcia-Roves, Aamir
R. Zuberi, Mohsen Karimi, Lea Rae Donahue, Derry C. Roopenian,
Jan Mulder, Mathias Uhlén, Tomas J. Ekström, Muriel T. Davisson,
Tomas Hökfelt, Martin Schalling, and Jeanette E. Johansen
High-throughput genotyping and quality control of gene-targeted
mutations in the mouse
Edward Ryder, Diane Gleeson, Debarati Bhattacharjee, Ross Cook and
the Mouse Genetics Programme
The Sanger Mouse Genetics Programme: High throughput
characterisation of immunological phenotypes in knockout mice
Mark Lucas, C Podrini, E Cambridge, S Clare, L Mottram, JK White
on behalf of the Mouse Genetics Programme
The Mouse Reference GENOME assembly: updates and resources
Valerie A Schneider and DM Church on behalf of the Genome
Reference Consortium and NCBI Genome Annotation Team
dbVar and dbSNP: NCBI Databases of Simple and Structural
Variations
Valerie A Schneider, Tim Hefferon, John Garner, Azat Mardanov,
Ming Ward, Aleksey Vinokurov, Melvin Quintos, Mike Kholodov,
David Shao, John Lopez, Steve Sherry, and Deanna Church
Current progress of phenotypic analysis in Japan mouse clinic
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Tomohiro Suzuki, Hideki Kaneda, Kimio Kobayashi, Ikuo Miura,
Tamio Furuse, Ikuko Yamada, Osamu Minowa, Hideaki Toki, Hiromi
Motegi, Maki Inoue, Tetsuo Noda, Hiroshi Maduya, and Sigeharu
Wakana
Hyperactive mice show a cluster of associated metabolic features
Jacqueline K White, Anna-Karin Gerdin, Christine Podrini on behalf
of the Mouse Genetics Programme, and Karen P Steel
Multiplicity of phenotypes in early generations of the mouse diversity
outcross population
Karen L Svenson and GA Churchill
SDOP-DB: a database for international sharing and standardization
of mouse phenotyping protocols
Nobuhiko Tanaka, Kazunori Waki, Hideki Kaneda, Tomohiro Suzuki,
Ikuko Yamada, Tamio Furuse, Kimio Kobayashi, Hiromi Motegi,
Hideaki Toki, Maki Inoue, Osamu Minowa, Tetsuo Noda, Shigeharu
Wakana, and Hiroshi Masuya
Modeling human chromosome 21-associated mental retardation
disorders in mice
Tao Yu, Zhongyou Li, Steven J Clapcote, Zhengping Jia, Chunhong
Liu, Annie Pao, Sei-ichi Matsui, Norma J Nowak, John C Roder, Chu
Chen, Allan Bradley, and Y. Eugene Yu
Taiwan Mouse Clinic – an experiment for mouse phenotyping
service
Jeffrey Jong-Young Yen, Yen-Hui Chen, and Ya-Wen Hsaio
Initial assembly of 17 mouse strains from the mouse genomes project
Guy Slater, Thomas M Keane, Jared Simpson, Aylwyn Scally,
Xiangchao Gan, Richard Mott, Jonathan Flint, and David J Adams
Clustered protocadherin genes: Genetic codes for generating the
complex brain
Takeshi Yagi, Shunsuke Toyoda, Yasushi Itoga, Keizo Hirano, Atsushi
Okayama, Shinichi Yokota, Shota Katori, Teruyoshi Hirayama, and
Takahiro Hirabayashi
Comprehensive phenotyping of mouse models
Tania Sorg, Marie-France Champy, Elodie Bedu, Roy Combe, Hamid
Meziane, and Yann Herault
Targeted and whole exome resequencing of mouse monogenic
mutants, quantitative trait loci and cancer models
David R. Beier, Jennifer L. Moran, Evan Mauceli, Snaevar
Sigurdsson, Tim Fennell, Lauren Ambrogio, Miriam H. Meisler,
John C. Schimenti, Karen Cichowski, Laura Reinholdt, Jane
Wilkinson, Stacey B. Gabriel, Federica di Palma, and Kerstin
Lindblad-Toh
Dynamic transcriptomes during neural differentiation of human
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embryonic stem cells revealed by short, long, and paired-end
sequencing
Jia Qian Wu, Lukas Habegger, Parinya Noisa, Anna Szekely, Caihong
Qiu, Stephen Hutchison, Debasish Raha, Michael Egholm, Haifan Lin,
Sherman Weissman, Wei Cui, Mark Gerstein, and Michael Snyder
Improving mouse genome annotation through new technologies
Laurens Wilming, Gary Saunders, Markus Brosch, Gavin Laird, Adam
Frankish, and Jennifer Harrow
Genomic alterations in tumors of the Mcm4Chaos3 breast cancer
mouse model
Marsha D. Wallace, Ethan Cerami, Stefan Stefanov, Lishuang Shen,
and John C. Schimenti
A missense mutation in the novel WD-repeat-containing protein,
WDR81, causes Purkinje cell and photoreceptor cell death in the
ENU-induced neurological mouse mutant nur5
Maria Traka, Kathleen J. Millen, and Brian Popko
Using whole genome next-generation sequencing as a tool to find
ENU-induced mutations
Michelle Simon, S Greenaway, P Denny, A-M Mallon, and JM
Hancock
The Sanger Mouse Genetics Programme; High throughput
morphological analysis of the eye
Valerie E. Vancollie, J Estabel, D Sunter, VB Mahajan, SH Tsang, MC
Naumann, M Mahajan, JK White, on behalf of the Mouse Genetics
Programme
A Cellular Genetics Platform to identify pharmacogenetic toxicity
pathways
O Suzuki, N Butz, M Pletcher, A Su, B Steffy, D Scoville, A Frick, J
Trask, R Thomas, and Tim Wiltshire
Lessons from phenotype of KO mouse, The role of Snur4 on
adipogenesis and insulin sensitivity
Jae Hoon Shin, Il Yong Kim, Yo Na Kim, Ji Won Choi, Kyung Jin
Roh, Mi Ra Sohn, Cheol soo Choi, Yun Soo Bae, and Je Kyung Seon
Epiblast-ablation of Sox2 expression leads to neurocristopathies in
mouse embryos
Nikolaos Mandalos, Marannia Saridaki, Jessica Lea Harper, Anastasia
Kotsoni, Aris N. Economides, and Eumorphia Remboutsika
A comprehensive catalogue of structural variation from! ! 17! ! inbred
mouse strains
Kim Wong, B Yalcin, T Keane, M Goodson, A Agam, C Nellaker, J
Stalker, J Flint, and D Adams
Collection and distribution of CRE/FLP-drivers at the Riken
Bioresource Center
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Atsushi Yoshiki, Kazuyuki Mekada, Hatsumi Nakata, Ayumi
Murakami, Masayo Kadota, Fumio Ike, Noriko Hiraiwa, Kaoru
Fukami-Kobayashi, Yuichi Obata
Analysis of the expression profile of CRH-POMC system genes in
vitiligo skin biopsies
Ene Reimann,, K Kingo, M Karelson, T Salum, E Aunin, P Reemann,
K Abram, E Vasar, H Silm, and S Kõks
Effects of a high spontaneous mutation rate in mammalian germline
by using mutator mice modified replicative DNA polymerase delta
Arikuni Uchimura, Yuko Hidaka, Ikuo Miura, Shigeharu Wakana, and
Takeshi Yagi
Tooling up MGI for a deluge in mouse phenotype and disease model
data
Anna Anagnostopoulos, Janan Eppig, and Mouse Genome Informatics
Group
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ABSTRACTS
35
ABSTRACT 1
THE F2 POPULATION FOR DISSECTING HOST SUSCEPTIBILITY
TO PERIODONTITIS
Ariel Shusterman1, Yael Houri-Haddad1, Ervin I Weiss1, and Fuad A Iraqi2
1
Department of Prosthodontics Hadassah Medical Center, Israel
2
Deptartment of Clinical Microbiology and Immunology, Sackler Faculty of
Medicine, Tel Aviv University, Israel
Introduction: Epidemiological studies in human have suggested that
susceptibility to periodontal disease is controlled by host genetic factors.
Recently, we have shown quantitatively by using microCT that inbred mouse
strains respond differently to the infection. Based on these results, we have
developed 408 mice of A/JxBALB/cJ F2 resource population, knowing that A/J
and BALB/cJ are the resistant and susceptible founder strains for the infection,
respectively.
Objective:
1) To determine the phenotypic response; i.e. alveolar bone loss, of all
progenies of the F2 population.
2) To perform genome wide search for quantitative trait loci (QTL) associated
with host susceptibility to periodontitis using selective genotyping approach.
Materials and Methods: 408 mice of the F2 population were generated by a
controlled mating scheme of the two parental inbred founders. All mice were
orally infected with Porphyromonas gingivalis and Fusobacterium nucleatum.
The infection was repeated three times at 2 day intervals. Six weeks following
the final infection, the maxillary jaws were harvested and alveolar bone loss was
quantified using microCT.
Results: The mean of phenotypic response of F2 population (0.0083mm3) was
intermediate between the two parental founders, 0.0103mm3 and 0.0062mm3 for
A/J and BABLB/cJ, respectively. The results suggest no genomic imprinting
(mitochondrial) effect as well as no significant sex effect.
Conclusions: The phenotypic variety of F2 population suggests more than one
quantitative trait loci (QTL) affecting host susceptibility to periodontitis. This is
the first study which uses mixed infection as experimental periodontitis that: (i)
compares different F2 groups (ii) analyze quantitatively a high number of mice
by microCT. Currently, we are finalizing the genotyping of 38% of the
phenotypic extremes of F2 resource population with 1500 single nucleotide
polymorphic (SNP) markers, covering the whole genome which allows
subsequently performing QTL mapping analysis. Mapping the QTL will point
candidate gene involved in periodontitis.
37
ABSTRACT 2
MOUSE
GENETIC
MODEL
OF
CARDIOVIRULENT
COXSACKIEVIRUS B3 INFECTION
Sean A. Wiltshire1,2 and Silvia M. Vidal1,2
1
Department of Human Genetics, McGill University
2
McGill Centre for the Study of Host Resistance, McGill University
In North America, up to 50% of Myocarditis can be attributed to Coxsackie
virus B3 (CVB3) infection. Host genetics, viral factors and the environment of
their interaction determine variable susceptibility to pathogenesis within
exposed human populations. Virally induced pathology in the mouse model
mimics the progression of disease in humans. Inbred mouse strains respond
differently to CVB3 infection including susceptible strains with A/J background
and resistant strains with C57BL/10 background. Through the analysis of a
segregating (A/JXB10.A)F2 cross we have recently detected three loci linked to
susceptibility to CVB3 induced pathology: Vms1, Vms2 and Vms3 (viral
myocarditis susceptibility). We have subsequently confirmed the existence of
Vms1 through the use of the consomic substitution strain CSS3 and the
localization of Vms1 through the analysis of a segregating (CSS3 x B6)F2 cross.
In order to identify causal genes we have undertaken a congenic and subcongenic approach to dissecting Vms1. Ongoing analysis of potential cell
autonomous mechanisms in isolated adult ventricular cardiomycytes may reveal
novel immune mechanisms which control infection in the heart.
38
ABSTRACT 3
ASSOCIATION
BETWEEN
SPATIAL
PROXIMITY
AND
FUNCTIONAL SIMILARITY IN HUMAN GENOME
Ekaterina Khraneeva, Andrey Mironov, and Mikhail Gelfand
Faculty of Bioengineering and Bioinformatics, M.V.Lomonosov Moscow State
University, Russia
A.A.Kharkevich Institute for Information Transmission Problems, RAS, Moscow,
Russia
Recent progress in determination of 3D structure of nuclear chromatin allows
one to study correlations between spatial proximity of genome domains and
their functional state. We combined the 3D data from (Lieberman-Aiden et al.
2009) with the results of several high-throughput studies of the chromatin
functional state. All pairs of regions from different chromosomes were divided
into groups according to their proximity, and the distribution of various
chromatin marks was calculated within these groups and then compared between
the groups. The results show that, indeed, gene regions that are spatially close
tend to have similar patterns of histone modifications (Encode 2004),
methylation state (Encode 2004), DNAse hypersensitivity (Encode 2004), and
expression level (Lieberman-Aiden et al. 2009). We also analyzed chimeric
transcripts as determined by genome mapping of paired-read RNA-Seq data (Xu
et al. 2010 and Berger et al. 2010) and observed that the frequency of pairs
mapping to two different genome loci is higher among spatially proximal
regions. This could be caused by (at least) two reasons: trans-splicing and
genome rearrangement compared to the reference genome (either somatic
recombination or allelic forms). To test these possibilities we analyzed the data
on genome trans-chromosomal rearrangements (Kidd et al. 2008) and
demonstrated that again the number of chimeric pairs is higher among spatially
close regions, although the effect is weaker than the one observed in the
transcriptomic data. Hence, there remains a possibility that the additional
chimeras indeed originate from trans-splicing. Finally, we analyzed GO
annotations of genes in spatially closest pairs of genome regions. The respective
sets were enriched for GO categories related to signal transduction and
regulation of transcription. In addition, pairs of spatially close regions had more
common GO terms than control random pairs. Taken together, these results
seem to demonstrate the presence of co-regulated genome domains formed by
regions of different chromosomes.
39
ABSTRACT 4
OLFACTORY RECEPTOR GENE FAMILY EVOLUTION IN
MARSUPIALS
Amir Mohammadi, Margaret L. Delbridge, and Jennifer A. Marshal Graves
ARC Centre of Excellence for Kangaroo Genomics and Research School of
Biology, The Australian National University, Canberra, Australia
The ability to smell is critical to most mammals, so it is not surprising that the
olfactory receptor genes are the largest gene family in genomes of eutherian
(placental) mammals. Pouch-bearing mammals (marsupials) diverged from
eutherian mammals about 148 million years ago (MYA), so we wished to
compare the size and diversity of this gene family between the two mammalian
infracalsses, as well as between an American and an Australian marsupial. The
complete genomes of two marsupials have now been sequenced, allowing us to
look at the conservation of large gene families in this group for the first time.
We therefore examined the composition of the olfactory receptor (OR) gene
family in the Brazilian short-tailed opossum (Monodelphis domestica) and the
tammar wallaby (Macropus eugenii), which diverged about 70 MYA. We
isolated all olfactory receptor gene sequences from the first assembly of the
Australian model marsupial, the tammar wallaby, classified them by
phylogenetic analysis and compared the distribution with the OR repertoire of
the opossum. Genomic location of most of ORG clusters was also identified in
tammar wallaby by using fluorescence in situ hybridization. Sequence
homology, and the genomic distribution of OR genes is well conserved between
these two distantly related marsupials. This high level of conservation contrasts
with the divergence of ORG families between eutherian mammals with similar
divergence times. Our results suggest a significant role for adaptive evolution,
rather than genomic drift, in the evolution of chemosensory genes in vertebrates.
40
ABSTRACT 5
ADVANCING PATERNAL AGE IS ASSOCIATED WITH DEFICITS IN
SOCIAL AND EXPLORATORY BEHAVIOURS IN THE OFFSPRING
Rebecca G. Smith1, Rachel L. Kember1, Leonard C. Schalkwyk1, Joseph D
Buxbaum2, Abraham Reichenberg1, Cathy Fernandes3*, and Jonathan Mill1*
1
Medical Research Council Social Genetic and Developmental Psychiatry
Centre, King's College London, London, United Kingdom
2
Department of Psychiatry, Mount Sinai School of Medicine, New York, New
York, United States of America
3
Department of Psychosis, King's College London, London, United Kingdom
*
Contributed equally to this work
Many epidemiological studies have demonstrated an association between
advanced paternal age and risk for several psychiatric disorders including
autism. In this study we used an animal model to investigate the effects of
advanced paternal age on behaviour in the offspring. C57BL/6J offspring were
bred from fathers of two different ages, two months old (to represent young
fathers) and 10 months old (to represent older fathers) using mothers aged two
months in both groups. The offspring underwent open field, holeboard and
social recognition tasks to explore differences in their behaviours. We observed
that the offspring of older fathers were found to engage in significantly less
social and exploratory behaviours than the offspring of younger fathers without
any reduction in their motor activity. Brain and peripheral tissues were obtained
from both parents and offspring and used to investigate de novo genomic
changes associated with increased paternal age using Nimblegen 720K CGH
microarrays. Spermatozoa undergo multiple divisions throughout the male
lifespan, potentially leading to a higher incidence of de novo copy number
variation (CNV) in any resulting offspring. Given recent human data reporting
an increased burden of CNVs in autism and other neurodevelopmental disorders,
we speculated that such genomic changes could be mediating the behavioural
changes we observed. Our study provides the strongest evidence for deleterious
effects of advancing paternal age on social and exploratory behaviour and
suggests that de-novo chromosomal changes are plausible explanatory factors.
41
ABSTRACT 6
GENOME-WIDE DIFFERENTIAL METHYLATION PATTERNS IN
INTERSUBSPECIFIC HYBRID MICE
John D. Calaway1,2,3,4, Hyuna Yang5, Elena de la Casa-Esperon7, Megan E.
Hanson6, Ezequiel C. Cambranis6, Maria A. Perez-Lamigueiro6, David L.
Aylor2, Leonard McMillan8, Gary A. Churchill5, and Fernando Pardo-Manuel
de Villena1,2,3,4
1
Curriculum in Genetics and Molecular Biology, University of North Carolina
University of North Carolina, Chapel Hill, NC
2
Department of Genetics, University of North Carolina, NC, USA
3
Lineberger Comprehensive Cancer Center, University of North Carolina, NC,
USA
4
Carolina Center for Genome Sciences, University of North Carolina, NC, USA
5
The Jackson Laboratory, Bar Harbor, ME, USA
6
Department of Biology, University of Texas Arlington, Arlington, TX, USA
7
Regional Center for Biomedical Research (C.R.I.B.) Albacete Science and
Technology Park/ University of Castilla-La Mancha, Spain
8
Department of Computer Science, University of North Carolina, NC, USA
Here we report the first genome-wide survey of the mouse differential
methylome. Using the high-density Mouse Diversity genotyping array, we
exploited the whole-genome amplification step of the Affymetrix genotyping
protocol by adding a pre-digestion step with either methylation-sensitive HpaII
or methylation-insensitive MspI endonucleases.
Reported previously as
methylation-sensitive single nucleotide polymorphism analysis (MSNP), this
technique compares genotyping calls between undigested and HpaII samples to
determine allele-specific methylation. We expanded the analysis by directly
analyzing probe intensity data to classify methylation patterns. By doing so, we
identified stably inherited epigenetic states attributed to parent-of-origin or
strain-of-origin. In addition, global methylation analysis suggests that female
mice exhibit a higher level of autosomal methylation when compared to males.
This approach identified three novel differential methylated regions (DMR).
We chose one located in the 3’ UTR of Actn1 for method validation using two
independent molecular assays. Our findings suggest that the mouse Actn1 DMR
has moderate tissue and strain specificity and a comparison of the orthologous
regions in rat and human reveal a possible rodent-specific DMR. Lastly, we
demonstrate that the Actn1 DMR influences gene expression based on
Collaborative Cross mice.
42
ABSTRACT 7
OPTIMIZATION OF A GENOME-WIDE, ALLELE-SPECIFIC DNA
METHYLATION ASSAY FOR THE MOUSE
John P. Didion1, John D. Calaway1, Isa Kemal Pakatci2, Leonard McMillan2,
and Fernando Pardo-Manuel de Villena1
1
Department of Genetics, Lineberger Comprehensive Cancer Center, Carolina
Center for Genome Science, University of North Carolina Chapel Hill, NC, USA
2
Department of Computer Science, University of North Carolina
Chapel Hill, NC, USA
Cytosine (CpG) methylation is involved in many critical cellular functions,
including genomic imprinting, Xchromosome inactivation and silencing of
retrotransposons and disease-related genes. Genomic studies increasingly call
for consideration of genome-wide methylation patterns, however the methods
available for assaying methylation in the mouse are currently limited either by
expense or the number and distribution of CpGs interrogated. This later
shortcoming is the result of most methylation studies focusing on CpG islands regions of high CpG concentration whose methylation status tends to be
correlated with the activity of nearby genes. However, recent studies have
shown that CpGs proximal to islands (as much as 4kb distant) are highly
correlated with allele-specific gene expression and thus of great interest to
researchers. We have optimized an existing method based on genotyping arrays
to assay allele-specific CpG methylation using the Mouse Diversity array and a
combination of methylation-sensitive and -insensitive restriction endonucleases.
Our method provides fully informative coverage of more than 100,000 CpGs,
and partially informative coverage of an additional 90,000 CpGs, most with at
least one technical replicate, and roughly even genomic distribution. We present
here a general bioinformatic approach for optimization of the Mouse Diversity
array to address a range of biological parameters. In addition, we have applied
our method to study parent-of-origin and strain-specific effects on methylation
in the Collaborative Cross, and have thus generated an optimized data set of
untreated controls that we provide as a community resource. Our findings will
reduce the expense and challenge of incorporating genome-wide methylation
analysis in genomic studies.
43
ABSTRACT 8
CHARACTERISATION
OF
LATERALITY
AND
CARDIAC
DEVELOPMENTAL DEFECTS IN THE K27 MUTANT MOUSE
Louise Stephen, K Mitchell, and K Hentges
University of Manchester, UK
Congenital heart defects account for 3% of all infant deaths and many more
prenatal. Cardiac development is a highly regulated process, reliant on the
cooperation of a vast number of genes. Whilst advances have been made in
defining the physiological processes occurring during cardiac development, the
genetic regulation is less well understood. Random mutagenesis screens offer a
phenotype-driven approach to producing novel mammalian models of human
congenital birth defects. We have applied this approach to the study of cardiac
development and are currently characterising the K27 mutant mouse, identified
from a balancer chromosome chemical mutagenesis screen. The K27 mutant
exhibits a recessive embryonic lethal phenotype, apparent from embryonic day
(E)9.5, with death occurring by E12. The K27 mutant exhibits a developmental
delay and abnormal cardiac morphology. Approximately 50% of mutants
demonstrate a reversal of cardiac looping. Analysis of Nkx2-5 and Fgf8 has
revealed that both the primary and secondary heart fields are correctly specified
in mutants. Likewise K27 mutants express Mef2c in a pattern indistinguishable
from control littermates, indicating that cardiac muscle is present in mutants.
Laterality defects suggested by the reversal in looping were confirmed by
investigation of the expression of genes required for left-right asymmetry,
including down-regulation of the Sonic hedgehog-regulated Gli1 gene in
mutants. Meiotic mapping has refined the K27 candidate region to a 5 Mb
interval on mouse Chromosome 11. This region contains over 100 genes, none
of which are currently known to have an established role in cardiac
development. Further work will include using high-throughput sequencing to
identify the K27 gene. Continued characterisation of the K27 mutants will
define the role the K27 gene plays in cardiac development and left-right
asymmetry. Combined these studies will identify a novel gene that contributes to
our understanding of cardiac congenital birth defects and of left-right axis
formation.
44
ABSTRACT 9
KYOTO EPILEPTIC RAT (KER): A NOVEL RAT MODEL OF KCNA1
MUTATION SHOWING SPONTANEOUS SEIZURES AND MUSCLE
INVOLUNTRY CONTRACTIONS
Saeko Ishida1, Tomoji Mashimo1, Takeshi Nishio2, Yu Sakamoto3, Shuji Kaneko 3,
and Tadao Serikawa1
1
Institute of Laboratory animals,and 2Department of Integrative Brain Science,
Graduate School of Medicine, 3 Department of Molecular Pharmacology,
Graduate School of Pharmaceutical Sciences, Kyoto University, Japan
Epilepsy is a neurological disorder characterized by unprovoked, recurring
seizures with a wide range of manifestations. We have so far performed
phenotype-driven ENU mutagenesis in rats to establish animal models of
epilepsy. Here we identified a rat exhibiting involuntary muscle movements and
convulsive seizures, named Kyoto Epileptic Rat (KER). We characterized KER
pathologically and physiologically and identified the responsible gene (Ker) for
involuntary muscle movements by positional cloning. KER was backcrossed by
more than 10 generations against F344/NSlc background strain to remove latent
ENU-induced mutations in other chromosomal regions. In KER, generalized
convulsive seizures were observed from 10 weeks of age, and most of the rats
died until 7 month of age. By electroencephalogram (EEG) recording,
polyspikes and polyspike-and-wave discharges were typically observed during
the seizures. By electromyogram (EMG) analysis of the anterior limb muscle, 7
Hz rhythmic spikes were detected during the interictal phase. Using 97 (KER x
BN/SsNSlc) x BN backcross progeny, Ker was mapped within the 10Mb region
of rat Chromosome 4. Within the region, a missense mutation T925A (S309T) in
voltage-gated potassium channel shaker-related subfamily member 1 (Kcna1)
gene was identified by sequencing analysis. The mutant homotetrameric Kv1.1
channels were non-functional in Xenopus oocytes. In humans, mutations of
KCNA1 cause autosomal dominant episodic ataxia type 1 (EA1) characterized
by brief episodes of ataxia associated with continuous myokymia. In addition,
the patients with EA1 are ten times more likely to develop epilepsy than normal
individuals. In mice, Kcna1 knockout mutation shows a recessive seizure
disorders (Smart et al., Neuron 1998), while V408A missense mutation shows
no seizures, but exhibits stress-induced loss of motor coordination (Herson et al.,
Nat Neurosci. 2003). Since KER shows muscular symptoms and spontaneous
seizures as an autosomal dominant manner, which are different from the
phenotype of the mouse models, KER can be a novel animal model of EA1 to
understand the underling mechanism of the KCNA1 diseases.
45
ABSTRACT 10
IDENTIFYING MODIFIER GENES OF MPNSTS IN THE NF1;P53CIS
MOUSE MODEL OF NEUROFIBROMATOSIS TYPE 1.
Jessica A Van Schaick1,2, Keiko Akagi3, Sandra Burkett1, Christina DiFabio1,
Robert Tuskan1, Jessica Walrath1, and Karlyne Reilly1
1
National Cancer Institute, Frederick, MD, USA
2
The George Washington University, Washington, DC, USA
3
The Ohio State University, Columbus, OH, USA
The current study aimed to identify modifier genes of malignant peripheral
nerve sheath tumors (MPNSTs) in the Nf1;p53cis (NPcis) mouse model of NF1.
Previous studies have shown that the incidence of MPNST development in the
NPcis mouse model is affected by the parental transmission of the mutant
Chromosome 11. In this study microarray analysis was used to examine gene
expression differences between MPNST primary tumors derived from NPcis
mice varying in inheritance of the NPcis chromosome from the mother (NPcis
maternal) or father (NPcis paternal). Grb10 was found to be more highly
expressed in NPcis maternal MPNSTs. Zrsr1 was found to be more highly
expressed in NPcis paternal MPNSTs. qPCR was used to validate both gene
expression differences. We chose to focus first on Grb10 due to its role as a
cytoplasmic signaling adapter protein. Fluorescence in situ hybridization was
used to examine the presence of Grb10 on Chromosome 11. Grb10 was found to
be lost more frequently in NPcis paternal MPNST cell lines, potentially
contributing to the decrease in Grb10 gene expression seen in these tumors.
Grb10 is paternally imprinted in the periphery of the mouse, therefore we
examined Grb10 isoform expression and found paternal and maternal isoforms
expressed in the MPNSTs. Due to these results we are examining whether loss
of imprinting is contributing to tumorigenesis. Finally we have generated
NPcis;Grb10cis mutant mice. Preliminary studies indicate that NPcis;Grb10cis
maternal mice have an increase in PNST incidence and a decrease in survival.
Our studies indicate that Grb10 is a candidate modifier gene of MPNSTs, acting
in a tumor suppressive manner.
46
ABSTRACT 11
A
NOVEL
GENETIC
MODEL
OF
OSTEOPOROSIS
BY
OVEREXPRESSION OF HUMAN RANKL IN TRANSGENIC MICE
Alexandra Niti1, R Dacquin2, V Rinotas1, P Jurdic2 and Eleni Douni1
1
Institute of Immunology, Biomedical Sciences Research Center “Alexander
Fleming”, Vari, Greece
2
Institut
de
Génomique
Fonctionnelle
de
Lyon,
UMR5242
CNRS/INRA/UCBL/ENS
Ecole Normale Supérieure de Lyon, France
Receptor Activator of Nuclear Factor-κB Ligand (RANKL, official symbol
TNFSF11) is a central regulator of bone remodelling by mediating osteoclastinduced bone resorption, whereas specific inhibition of RANKL by a fully
human monoclonal antibody effectively reduces the incidence of fractures in
postmenopausal women. We have recently generated transgenic mice
overexpressing human RANKL (TghuRANKL) in order to model human
RANKL-mediated pathologies. To achieve a correct pattern of human RANKL
expression in the mouse, a transgenic 200kb genomic fragment containing the
whole human RANKL gene has been introduced into the mouse genome. Among
the five independent transgenic lines obtained, a striking skeletal phenotype was
observed in the highest copy number TghuRANKL founder characterized by
multiple bone fractures, increased osteoclast numbers, severe trabecular bone
loss and cortical porosity. A similar but milder bone phenotype was identified
by another high copy TghuRANKL line that developed both trabecular bone
loss and cortical bone porosity by the age of 3 months. Additionally, a low copy
number TghuRANKL line developed exclusively trabecular bone loss by the age
of 3 months. The observed phenotypes in various TghuRANKL lines developed
in both sexes, whereas the levels of human RANKL expression were correlated
with disease severity. Notably, the osteopetrotic phenotype of mutant mice
expressing an inactive form of the endogenous RANKL protein was rescued
upon crossing these mice with TghuRANKL mice, showing that the transgenic
human RANKL protein is fully active in the mouse. These novel human
RANKL transgenic mice represent a unique tool for understanding the
pathogenic mechanisms that cause bone resorption and for the evaluation of
novel therapeutic approaches targeting RANKL-mediated pathologies such as
osteoporosis.
47
ABSTRACT 12
POSITIVE SELECTION MIGHT CONTRIBUTE TO FUNCTIONAL
DIVERGENCE OF NAIPS FOLLOWING GENE DUPLICATION IN
MAMMALS DURING THE EVOLUTION
Zhaoliang Su1, Chenglin Zhou4, Jianguo Chen2*, Shengjun Wang1*, and Huaxi
Xu1*
1
Department of Immunology & Laboratory Immunology, Jiangsu University,
Zhenjiang, China
2
The Affiliated People’s Hospital of Jiangsu University, Zhenjiang, China
3
Zhenjiang Entry-exit Inspection and Quarantine Bureau, Jiangsu, China
The neuronal apoptosis inhibitor proteins (NAIPs) are members of NLRs (Nodlike receptors) protein family. Recent researches have shown that some NAIP
genes were strongly associated with both mammalian innate immunity and many
diseases, whereas some NAIP genes had been pseudogenization. So the present
works were to clarify whether the functional divergence had appeared, as well as
whether natural selection played an important role during their evolution. Our
results showed that NAIP genes had produced functional divergence and
appeared genes duplicated before the divergence; during evolution, lineagespecific duplications of NAIP genes were gradually replaced by
pseudogenization and NAIP genes were driven by the positive selection; and the
selective force changed the functional sites of NAIP, affected the tertiary
structure of NAIP, and then produced the functional divergence. Our results also
indicated that the positive selection and functional divergence of NAIP might
provide mammals advantages to adapt evolution of geographical environment.
48
ABSTRACT 13
A FORWARD GENETICS APPROACH IDENTIFIES A NOVEL
MEMBER
OF
THE
DNAJC
FAMILY
THAT
CAUSES
NEUROMUSCULAR DISEASE IN MICE
Fotios Ioakeimidis, Vagelis Rinotas, Eleni Makrinou, George Kollias, and
Eleni Douni
Biomedical Sciences Research Center “Alexander Fleming”, Vari, Greece
Neuromuscular diseases encompass a wide range of clinical conditions
remaining incurable while the genetic and molecular basis of most of these
conditions remains unknown. Using mouse ENU mutagenesis we have
identified a novel autosomal recessive neuromuscular phenotype. This
phenotype manifests with hind limb weakness, progressing with muscle atrophy,
reduced body weight, generalized paralysis and death within 30 days after birth.
Genetic linkage analysis mapped the causal mutation within a 2Mbp interval
containing 24 genes. Sequencing revealed an intronic T>A transversion within a
member of the DnaJ homolog, subfamily C (DNAJC) of heat shock proteins
(Hsp40s). This is a novel gene with completely unknown function, whose
protein is suggested to be located in the mitochondrion, suggestion that is
supported by our own confocal imaging data. The intronic mutation generates a
novel splicing acceptor site resulting in the insertion of 109 bp in the mature
transcript, causing a frameshift of the last 50 aminoacids of the protein that are
replaced by 44 different aminoacids. This DNAJC family member shares 96%
identity with its human ortholog, highlighting the functional importance of the
protein. Our expression analysis data show constitutive and high expression of
the normal gene in all tissues studied, neuronal and non neuronal. To genetically
prove the causality of this mutation in our phenotype, we have generated BAC
transgenic mice for the human DNAJC gene in order to rescue the
neuromuscular phenotype. Our current studies focus on the histological and
immunohistochemical characterization of the CNS in our mutant mice, and
analysis of the expression pattern and function of the normal and the mutated
protein. The identification of a novel DNAJC protein involved in neuromuscular
disease and future functional characterization of this protein can shed light into
new pathogenetic pathways involved in neuromuscular diseases.
49
ABSTRACT 14
GENE EXPRESSION STUDIES OF ncRNAS WITHIN THE ODZ4
LOCUS
Tyler Buit, Chiao-Ling Lo, Feichen Shen, and Amy C. Lossie
Department of Animal Sciences, Purdue University, West Lafayette, IN, USA
The Odz4 allelic series is comprised of five phenotypically distinct embryonic
lethal mouse mutants, which are characterized by abnormal embryonic and
extraembryonic mesoderm development, irregular somite formation, imperfect
patterning of the skeleton and defective maternal blood flow to the embryo.
Odz4 gene regulation is complicated and not well understood. Whole
transcriptome and cross-species comparison studies suggest that the 735 kb
locus produces over 20 alternatively spliced transcripts, contains over 800
CAGE tags and includes 93 highly conserved non-coding elements (HCEs).
These HCEs are >200bp long and exhibit ≥70% identity among five mammalian
species. Similar highly-conserved DNA sequences can contain mRNA-like noncoding RNAs that control developmental gene expression. We hypothesize that
several of these HCEs will encode cis-acting ncRNAs that direct alternative
splicing at the Odz4 locus. Our long-term goal is to determine the role of these
HCEs in directing Odz4 transcription during development. Using RT-PCR, we
demonstrated that 92 of the 93 HCEs are expressed in the adult and/or at various
embryonic stages in normal embryos. Each transcript exhibited a unique
developmental expression profile. One HCE was detected at the blastocyst stage,
which is before the primary Odz4 transcripts are produced. Six HCEs were
detected in E6.5 embryos, an important stage in ODZ4 signaling. Northern and
RT-PCR analyses indicate that although several of the ncRNAs are new Odz4
exons, at least 60 of these HCEs are expressed sequences of unknown function,
all of which lie within 5 kb of an Odz4 CAGE tag. In situ hybridization studies
at later time points indicate that these six HCEs demonstrate dynamic expression
profiles. For example, at E9.5, Hce39 is restricted to punctate regions in the
limbbud, brain and first brachial arch. By E12.5, we detect broad expression
across the neural ectoderm, with high-levels at the midbrain-hindbrain junction.
RT-PCR and in situ hybridization studies of the HCEs in Odz4 mutant embryos
is ongoing, and future studies are aimed at determining the effects of these
ncRNAs in specific Odz4 mutants.
50
ABSTRACT 15
EARLY LIFE STRESS IS ASSOCIATED WITH ALTERED
BEHAVIOUR AND EPIGENETIC ALTERATIONS TO NR3C1, AVP
AND NR4A1
Rachel L. Kember, Emma Dempster, Leonard C. Schalkwyk, Jonathan Mill, and
Cathy Fernandes
Institute of Psychiatry, Kings College London, UK
Stressful early life environments have consistently been implicated in the
development of adverse behavioural phenotypes. Environmental effects can be
mediated by epigenetic processes, whereby changes in epigenetic marks due to
environment can produce long lasting changes to phenotype. A mouse model is
an ideal way to disentangle the contributions of environment, genetics and
epigenetics towards phenotypic outcome, as it allows both the genetic and the
environmental contribution to be controlled. The current study investigates the
behavioural effects of a model of early life stress (maternal separation) in mouse
using two inbred strains (C57BL/6J and DBA/2J). A battery of behavioural tests
demonstrates that the maternal separation group exhibit phenotypic differences
when compared to controls, including variation in anxiety levels. Corticosterone
levels, measured at baseline and post-forced swim test, show increased hormone
levels in C57BL/6J maternally separated animals after the forced swim. We
identified three candidate genes that have previously shown associations with
early life stress. Epigenetic changes in the glucocorticoid receptor (Nr3c1) and
arginine vasopressin (Avp) genes have both been associated with poor maternal
behaviour and maternal separation, respectively. Additionally, nerve growth
factor IB (Nr4a1) is rapidly induced by various stress responses as an early
response mechanism. I will present findings that show significant methylation
differences in the hippocampus of maternally separated animals compared to
controls at CpG sites in the promoter regions of these genes. Furthermore,
preliminary data from a genomewide methylation assay will be presented.
Studies such as these will allow further disentanglement of environment,
genotype and epigenetic mechanisms in the contribution towards adverse
phenotypes.
51
ABSTRACT 16
STRESS MECHANISMS, INFLAMMATION, INFECTION AND HOST
RESISTANCE
Katia P. Karalis
Biomedical Research Foundation of the Academy of Athens (BRFAA), Greece
and Children’s Hospital, Harvard Medical School, Boston, USA
Stress response, a process well-preserved in mammals, is mediated by the
activated hypothalamic-pituitary-adrenal (HPA) axis. Immune system
challenges such as inflammatory and infectious stimuli activate stress response
leading to release of glucocorticoid. The latter is a very potent
immunosuppressive and anti-inflammatory agent when administered
exogenously. Glucocorticoid acts through binding to glucocorticoid receptor
(GR), two types α and β have been identified in humans with antagonistic
effects in some cases. In addition to the GRs abundance, the local effects of
glucocorticoid are regulated by the expression of 11betaHSD, an enzyme that
modulates the availability of biologically active levels of glucocorticoid, in a
tissue-specific manner. The hypothalamic mediator of the stress response,
Corticotropin Releasing Hormone or Factor (CRH/CRF), is also expressed in
various peripheral tissues and exerts potent immunomodulatory effects. This
factor belongs to a family of peptides acting, although with different affinity, via
two G-protein coupled receptors (CRF receptor 1 and 2) expressed in several
sites in the nervous system and in a plethora of peripheral tissues. Studies from
our and other laboratories have demonstrated the bidirectional, direct and
indirect, effects of CRF in inflammatory processes and have started to elucidate
the mechanisms mediating these actions of CRF. In this presentation we will
review the evidence that supports the critical role of the, so-called, stress
hormones in the resistance to inflammatory challenges. Next, we will present
our findings from mouse models demonstrating a dual role of the stress system
in host resistance and the molecular pathways implicated. Finally, we will
discuss the possibilities for novel therapeutic applications for inflammatory
conditions, given the challenges provided by the complex regulation of the
genes involved in the stress response.
52
ABSTRACT 17
PHYSIOLOGIC CONSEQUENCES OF CONSTITUTIVE MTOR
INHIBITION: ALTERATIONS IN CELL/ORGAN SIZE AND IMMUNE
CELL DEVELOPMENT
Shuling Zhang1, Julie A. Readinger2, Wendy DuBois1, Mirkka Janka-Junttila3,
Richard Robinson1, Magaret Pruitt1, Val Bliskovsky1, Julie Z. Wu2, Kaori
Sakakibara1, Jyoti Patel1, Carole A. Parent3, Lino Tessarollo4, Pamela L.
Schwartzberg2, and Beverly A. Mock1
1
Laboratory of Cancer Biology and Genetics, CCR/NCI, 2Genetic Disease
Research Branch, NHGRI, and 3Laboratory of Cellular and Molecular Biology,
CCR/NCI, 4Mouse Cancer Genetics Program, CCR/NCI
Mammalian TOR (MTOR) regulates cell growth, proliferation and migration.
Because Mtor knock-outs are embryonic lethal, we generated a hypomorphic
mouse with a neo-insertion that partially disrupts MTOR transcription and
reduces its expression, thus creating a physiologic model of TORC1/TORC2
inhibition. Homozygous knock-in mice exhibited reductions in body, organ and
cell size. Although reductions in most organ sizes were proportional to
decreased body weight, spleens were disproportionately smaller. Decreases in
the total number of T cells, particularly memory cells, and reduced responses to
chemokines suggested alterations in T cell homing/homeostasis.
TCRstimulated T cells also proliferated less, produced lower cytokine levels, and
induced an increased number of regulatory T cells (iTreg). Surprisingly, B cell
developmental effects were more pronounced, with a partial block in B cell
development in bone marrow, altered splenic populations, decreased migration
to chemokines, and decreased proliferation. Moreover, AKT phosphorylation
was increased in activated B cells, reminiscent of cancers treated with
rapamycin. Furthermore, decreased neutrophil numbers were observed in the
spleen, despite normal development and migration in the bone marrow. Thus,
MTOR is required for the maturation and differentiation of multiple immune
cell lineages. These mice provide a novel platform for studying consequences
of constitutively reduced TORC1/TORC2 activity.
53
ABSTRACT 18
SEVERE
COMBINED
IMMUNODEFICIENCY
(SCID)
RATS
GENERATED BY ZINC FINGER NUCLEASE TECHNOLOGY
Tomoji Mashimo, Akiko Takizawa, Birger Voigt, Kazuto Yoshimi, Hiroshi Hiai,
Takashi Kuramoto, and Tadao Serikawa
Institute of Laboratory Animals, Graduate School of Medicine, Kyoto
University, Kyoto, Japan
Although the laboratory rat has been widely used as animal models across many
fields in biomedical sciences, the inability to utilize germline-competent
embryonic stem (ES) cells was a major drawback for research activities that
aimed to elucidate gene functions. Recently, Zinc-Finger Nucleases (ZFNs)
have been used successfully to create site-specific DNA double-strand breaks,
and thereby stimulate targeted gene mutations in a wide variety of organisms
including plants, xenopus, drosophila, zebrafish, and rats. Here we demonstrate
ZFN-stimulated gene-targeting at an endogenous rat gene, for which human and
mouse mutations are known to cause a severe combined immunodeficiency
(SCID). Co-injection of mRNAs encoding the custom-designed ZFNs into
pronucleus of fertilized oocytes yielded 25% gene-modified offspring, including
a wide variety of deletion or insertion mutations. ZFN-modified founders
faithfully transmit these genetic changes to the next generation with the SCID
phenotypes. The high frequency of gene-targeting and the rapid creation of gene
knockouts indicate that ZFN technology can provide a new strategy in rats for
creating animal models of human diseases.
54
ABSTRACT 19
A
RECOMBINATION
HOTSPOT
LEADS
TO
SEQUENCE
VARIABILITY WITHIN A NOVEL GENE AND CONTRIBUTES TO
COMPLEX DISEASE SUSCEPTIBILITY
Iris KL Tan, Leanne Mackin1, Nancy Wang1,2, Anthony T Papenfuss3, Colleen M
Elso1, Michelle P Ashton, Belinda Phipson2,3, Melanie Bahlo3, Terrence P
Speed3, Gordon K Smyth3, Grant Morahan4, and Thomas C Brodnicki1
1
St Vincent’s Institute of Medical Research, Fitzroy, Victoria, Australia
2
The Walter & Eliza Hall Institute of Medical Research, Parkville, Victoria,
Australia
3
The Western Australian Institute of Medical Research, Perth, Western
Australia, Australia
Type 1 diabetes (T1D) is a multigenic autoimmune disease in which
lymphocytes mediate destruction of insulin-producing pancreatic beta cells.
Despite the recent success of human genome-wide association studies, genetic
heterogeneity and tissue availability still hinder the identification of causative
alleles, as well as their effects upon gene function and T1D pathogenesis. A
synergistic approach to ongoing human studies is the use of the nonobese
diabetic (NOD) mouse strain, which has provided key insights into T1D. We
have recently positionally cloned a T1D susceptibility locus, termed Idd11,
located on mouse Chromosome 4. Sequence analysis across a critical 6.9 kb
interval in a series of congenic NOD mouse strains and in 25 other inbred strains
identified several haplotypes, including a unique NOD haplotype, associated
with varying levels of T1D susceptibility. Haplotype diversity within this
interval between congenic NOD mouse strains was due to a recombination
hotspot that generated four crossover breakpoints, including one with a complex
conversion tract. The Idd11 haplotype and recombination hotspot are located
within a predicted gene of unknown function. This gene, temporarily named
Latet, exhibits deficient splicing and decreased expression in relevant
immunological tissues of NOD mice. Notably, it was the recombination hotspot
that aided our mapping of Idd11 and confirms that recombination hotspots can
create genetic variation representing ‘private mutations’ and affecting a common
polygenic disease. This finding has implications for studies of complex genetic
disease, which may be affected by the ~33,000 estimated recombination
hotspots in the human genome.
55
ABSTRACT 20
A SENSITIZED SUPPRESSOR SCREEN TO IDENTIFY MODIFIER
GENES FOR FACTOR V LEIDEN-DEPENDENT THROMBOSIS IN
THE MOUSE
Randal Westrick, Goujing Zhu, Sara Manning, Angela Yang, David Siemieniak,
and David Ginsburg
University of Michigan, Ann Arbor MI USA
Venous thrombosis affects ~300,000 individuals/year in the USA. A factor V
polymorphism, Factor V Leiden, (FVL) is the most common genetic risk factor
for venous thrombosis, though it displays only 10% penetrance. We previously
demonstrated synthetic lethality between homozygosity for FVL (FVQ/Q) and
heterozygous tissue factor pathway inhibitor deficiency (TFPI+/-) in mice. To
identify modifier genes contributing to FVL penetrance, we used this lethal
phenotype for a sensitized ENU mutagenesis screen. As proof of concept, we
tested the loss of one tissue factor (TF+/-) allele to suppress the lethal FVQ/Q
TFPI+/- phenotype. Analysis of 257 offspring from a FVQ/Q X FVQ/+ TFPI+/TF+/- cross demonstrated that haploinsufficiency for TF suppressed FVQ/Q
TFPI+/-. Thus, suppressor mutations in the TF gene should emerge from our
screen. We next performed a genome-wide mutagenesis screen for suppressors
of FVQ/Q TFPI+/-. Male FVQ/Q mice were exposed to ENU and bred to FVQ/+
TFPI+/- females. Surviving G1 offspring were analyzed to identify mice with the
otherwise lethal FVQ/Q TFPI+/- genotype. Analysis of 7,128 G1offspring (~2X
genome coverage) identified 98 FVQ/Q TFPI+/- mice that survived to weaning. 13
FVQ/Q TFPI+/- G1 mice exhibited successful transmission of a putative
suppressor mutation to two or more FVQ/Q TFPI+/- G2 offspring. 11 of these 13
putative suppressors are being subjected to positional cloning to identify the
corresponding mutations. Analysis of 20 offspring from one of the lines
resulted in the identification of a Chromosome 3 region encompassing the TF
gene (LOD=4.93). Sequencing of this region is in progress to identify the
causative mutation. Mapping and genome sequencing is underway for the
remaining 10 lines. Identification of these mutations should provide novel
insights into hemostatic regulation and suggest candidate modifier genes for
Factor V Leiden and other human hemostatic disorders.
56
ABSTRACT 21
GENETIC AND DIETARY CONTROLS OF ALTERNATIVE EXON USE
AND MRNA LEVELS IN THE SYSTEMS ARCHITECTURE OF DIETINDUCED METABOLIC DISEASES
Sabrina H. Spiezio1, Annie Hill-Baskin1, Karen Fitch2, Keith Jones2, Michael H.
Shapero2, and Joseph H. Nadeau1
1
Department of Genetics, Case Western Reserve University, Cleveland, Ohio,
USA
2
Affymetrix Inc., Santa Clara, CA, USA
Because of their rapidly increasing incidence worldwide, the need is urgent to
characterize the genetics and systems biology of obesity and related metabolic
conditions such as metabolic syndrome, type 2 diabetes, and non-alcoholic fatty
liver disease (NAFLD). Two mouse inbred strains, C57BL/6J (B6) and A/J,
show contrasting metabolic responses to high fat vs low fat diets. Using a
complete panel of chromosome substitution strains (CSSs) that we made to
study genetically complex traits such as these, we discovered 100s of complex
trait genes that have unexpectedly large and non-additive phenotypic effects.
Moreover these networks of interacting genes buffer systems properties from
genetic perturbations. To examine molecular features of these systems, we
compared mRNA and alternative exon profiles in three metabolically relevant
tissues, for two diets, and several CSSs and B6 control mice that differ in their
response to high fat vs low fat diet. The timepoint was midway through the
study period, after 50 days on the test or control diet, but before significant
weight gain was evident. Key findings include 103 more mRNAs showing dietinduced differential expression and 104 more diet-induced alternative exon
usages in CSSs than B6, cis-effects that were more common than trans-effects
on the low fat vs. high fat diet, remarkable heterogeneity in trans-responses to
different chromosome substitutions, and finally a modest correlation between
changes in mRNA expression and use of alternative exons. Together, these
results suggest that changes in alternative exon use are much more common than
changes in mRNA levels, that B6 responded in a markedly different molecular
manner than CSSs to dietary perturbations, and that alternative exon use may be
an important but generally neglected source of functional heterogeneity for
modulating molecular and physiological responses to genetic and dietary
perturbations.
57
ABSTRACT 22
A GENE DRIVEN ENU MUTAGENESIS SCREEN REVEALS A NOVEL
METHOD OF ZIC PROTEIN NUCLEAR LOCALIZATION
R Ali1, H Bellchambers1, N Warr2, D Quwailid2, P Denny2 and Ruth Arkell1,2
1
Early Mammalian Development, Research School of Biological Sciences, The
Australian National University, Canberra, ACT, Australia
2
Mammalian Genetics Unit, MRC Harwell, Oxfordshire, UK
The Zic genes are an important family of transcription factors about which little
is known. Germ line mutation of Zic genes leads to a variety of congenital
defects and somatic mutation of ZICs is implicated in a wide range of cancers.
The Zic genes contain a zinc finger domain with supposed DNA binding, protein
binding and nuclear localization roles. To learn more about the specific
molecular functions of the zinc finger domain we undertook a search for
relevant point mutations within the Harwell archive of mutagenised mouse
DNAs. The Zic5 gene screen lead to the isolation of 2 missense alleles, one with
a conservative amino acid change and one with a non-conservative change. In
cell based transcription assays both mutant proteins exhibit a partial loss-offunction and mouse strains harbouring theses mutations have a milder phenotype
than a targeted null allele of Zic5. Together this demonstrates that both
mutations have generated hypomorphic alleles of Zic5. We have further
investigated how a conservative change causes a partial loss-of function. The
missense mutation alters a lysine residue and the lysine is a predicted substrate
for SUMOylation. In vivo studies indicate that Zic5 is normally SUMOylated at
this and other lysines and that this modification is required for localization to the
nucleus. Other ZIC proteins have been shown to be trafficked to the nucleus via
the importin pathway. Our data therefore point to a previously unrecognized
mode of transport for the ZIC proteins. SUMOylation is a post-translational
modification that is frequently associated with transcription factors. Previous
work has demonstrated that the absence of all SUMOylation is incompatible
with development of an organism, however, the effect on individual proteins has
been harder to demonstrate. This work provides the first in vivo evidence that
SUMOylation is required for the function of an individual protein.
58
ABSTRACT 23
NON-MUSCLE MYOSIN IIB IS REQUIRED FOR FORMATION OF
THE CORONARY VASCULATURE
Karen Mitchell, C Clowes, Louise Stephen, and Kathryn E Hentges
Faculty of Life Sciences, University of Manchester, Manchester UK
The coronary vasculature is an essential network providing the blood supply to
the heart. Disruptions in the coronary circulation deprive the working
myocardium of oxygen, leading to irreversible damage to cardiac muscle and
myocardial infarction. Cardiovascular disease is a major contributor to
mortality worldwide. The generation of treatments for cardiovascular disease
will be aided by a deeper understanding of the developmental processes
underlying the formation of the coronary vessels. We have identified a
requirement for Non-Muscle Myosin IIB (NMIIB) in the development of the
coronary vasculature. We isolated an embryonic lethal mouse mutant with
Embryonic Hydrocephalus and Cardiac defects (EHC) from a balancer
chromosome mutagenesis screen. Positional cloning revealed that the EHC
mutant phenotype is caused by a splice donor mutation in Myh10, which
encodes NMIIB. EHC mutants produce an aberrant transcript lacking exon 18,
disrupting the reading frame of NMIIB and introducing a premature stop codon.
RT-PCR analysis demonstrates that the mutation destabilises the Myh10
transcript, such that heterozygous embryos preferentially produce wild type
Myh10. EHC mutant embryos display cardiac morphological defects such as
double-outlet right ventricle. Additionally mutants do not form coronary
vessels, evident from gross inspection of hearts and a lack of vascular
endothelial PECAM staining. The failure to form the coronary vasculature is
suggestive of epicardial defects. Accordingly, EHC mutant epicardial cells fail
to form an epithelial layer covering the myocardium, and have disruptions in the
localisation of extracellular matrix between the myocardium and epicardium.
NMIIB therefore may facilitate coronary vessel formation by supporting
adhesive interactions between the epicardium and myocardium mediated via the
extracellular matrix. Future work will determine the mechanisms by which
Myh10 deficiency contributes to coronary vascular failure.
59
ABSTRACT 24
SHORT CIRCUIT, A HOMOZYGOUS LETHAL ENU MUTATION
THAT AFFECTS LUNG DEVELOPMENT
Michael J. Parsons, Laura Yates, Jessica Edwards, Lauren Chessum, Charlotte
H. Dean, and Patrick M. Nolan
Mammalian Genetics Unit, MRC Harwell, Oxfordshire, United Kingdom
The use of N-ethyl-N-nitrosourea (ENU) mutagenesis screening is a powerful
tool for discovering novel genes associated with numerous biological pathways
and phenotypes. The mutant called short circuit (Sci) was found in a dominant
enu mutagenesis screen. This mutant, first characterized in a screen for
circadian phenotypes, was found to be homozygous lethal with Sci homozygous
animals dying between embryonic day 14.5 to post-natal day one. The gene
containing the Sci mutation encodes for a protein that is a transcription factor
with multiple zinc fingers. This gene has been implicated in various functions
including neuronal differentiation and development. In mouse, this gene is
highly expressed in developing brain, lung and heart, with its expression
peaking at embryonic day 13.5. These expression patterns combined with the
timing of the lethality in homozygous animals led us to explore the possibility of
the Sci mutation interfering with normal lung development, thus leading to the
observed homozygous lethality. Upon conducting a time series of lung
development, we found significant decreases in distal airway volume in Sci
homozygous animals, as compared to wildtype animals, at embryonic day 18.5
(p<0.05); morphological changes in distal airways were observed as early as
embryonic day 15.5. Adult heterozygous Sci animals showed impaired lung
function as measured by full body plethysmography following metacholine
challenge. Specifically, heterozygous Sci animals had significant decreases in
baseline PenH (a measure of lung resistance), peak inspiratory flow and peak
expiratory flow (p<0.05). We are currently conducting immunohistochemical
analysis using lung differentiation and proliferation markers, to further
characterize the effects of this gene on lung development. Taken together these
data suggest that this gene may play an important role in lung development.
60
ABSTRACT 25
IDENTIFICATION OF THE DANFORTH’S SHORT TAIL MUTATION
USING NEXT GENERATION SEQUENCING
Christopher N. Vlangos, Amanda N. Siuniak, Dan Robinson, Arul M.
Chinnaiyan, James Cavalcoli, Robert H. Lyons, and Catherine E. Keegan
University of Michigan, Ann Arbor, MI USA
The Danforth’s short tail (SdSd) mouse first appeared as a semi-dominant
spontaneous mutation in an inbred colony at Stanford University in the 1920s.
The phenotype of heterozygous (SdSd/+) animals includes unilateral kidney
agenesis, vertebral anomalies, and a shortened and kinked tail. Homozygous
(SdSd/Sd) mice are more severely affected with bilateral renal agenesis, lack of
tail, vertebral anomalies, spina bifida, lack of urogenital and anal openings, and
persistence of the cloaca. Homozygous mice are born live but die within 24
hours of birth. Though genetically mapped to a 1.0cM (1.5Mb) region of mouse
Chromosome 2qA3, the Sd mutation has not yet been identified. Using
bioinformatic analysis of the mouse DNA physical map we identified 9
annotated genes with a total of 86 coding exons spanning the corresponding
1.5Mb critical region. Direct sequencing of the exonic DNA and intron/exon
boundaries did not reveal any causative mutations. Since direct sequencing of
the exonic DNA only provided ~1% coverage of the Sd critical region we
performed next generation sequencing (NGS) of the entire 1.5Mb region. Prior
to NGS we performed an enrichment capture using an Agilent oligo DNA chip
designed to cover all unique (non-repeat masked) DNA of the Sd critical region.
After successful locus specific DNA enrichment, 36bp paired end NGS on an
Illumina Genome Analyzer IIx was completed, generating 1.85Gb of sequence.
93% of reads were of proper size and mapped to the enriched region in the
proper orientation resulting in an average of 200X coverage. Analysis of the
mapped reads using standard computational techniques did not reveal any
causative mutations. We then turned to interrogation of reads where only a
single end of the paired end sequencing mapped correctly to the Sd locus. By
using this novel technique, we were able to identify the presence of an
endogenous retroviral-like (ERV) insertion of the MusD family at the Sd locus.
The ERV insertion was confirmed via Southern analysis, and is not present in
additional inbred mouse lines tested. The phenotypic characteristics of
homozygous SdSd/Sd mice are comparable to those seen in human patients with
caudal regression syndrome, cloacal exstrophy, and VACTERL association.
Thus, the Sd mouse is an excellent model for identification of novel genes
responsible for human disorders.
61
ABSTRACT 26
A NOVEL ROLE FOR ATMIN, CONTROLLING CILIOGENESIS
THROUGH MODULATION OF DYNEIN LIGHT CHAIN EXPRESSION
Paraskevi Goggolidou1, Jonathan Stevens1, Gabrielle Wheway3, Antonella Di
Paolo, Rosario Romero1, James Briscoe2, Colin Johnson3, and Dominic P
Norris
1
Mammalian Genetics Unit, Medical Research Council Harwell, Oxfordshire
OX11 0RD, UK
2
National Institute for Medical Research, London, UK,
3
Section of Ophthalmology and Neurosciences, Wellcome Trust Brenner
Building, Leeds Institute of Molecular Medicine, St James's University Hospital,
Beckett Street, Leeds, U.K.
Through a phenotype driven genetic screen, we identified the DNA damage
response locus Atmin as encoding a zinc finger factor required for normal
ciliogenesis. Point mutants destroying Atmin zinc finger structure result in
embryonic death around 13.5 days of development. Homozygous mutants
exhibit a complex developmental syndrome indicative of a ciliopathy, including
exencephaly, coloboma, cardiac outflow tract defects, pulmonary hypoplasia
and left-right (L-R) patterning defects. Indeed, impaired ciliogenesis is evident
from a decreased number of ciliated cells and shortened cilia in the embryonic
node. Notably, these morphologically abnormal cilia retain motility, although LR patterning is abnormal. Cilial structure is known to be important for SHH
signaling and for correct establishment of dorso-ventral (D-V) patterning in the
neural tube. A defect in SHH signalling is evident in Atmin mutants, as assessed
by GLI3 processing. However, no obvious defects in neural tube D-V patterning
were detected, suggesting a specific role of Atmin in GLI3 but not GLI2
processing. We have further identified a dynein light chain as acting
downstream of Atmin; mRNA expression analysis shows a ~16-fold
downregulation in Atmin mutant embryos. Using si-RNA mediated knock down
of Atmin and the dynein, we show that Atmin mediates ciliogenesis through
regulation of expression of this specific dynein subunit. Sub-cellular localisation
places this dynein subunit in the primary cilium in both cultured cells and the
embryo, consistent with it directly influencing retrograde intraflagellar transport.
62
ABSTRACT 27
GENETIC ANALYSIS OF COMPLEX TRAITS IN THE EMERGING
COLLABORATIVE CROSS
David L. Aylor1, William Valdar1,#, Wendy Foulds-Mathes1,#, Ryan J. Buus1,#,
Ricardo A. Verdugo2,#, Ralph S. Baric3,4, Martin T. Ferris1, Jeffrey A.
Frelinger4, Mark Heise1, Matt B. Frieman4, Lisa E. Gralinski3, Timothy A. Bell1,
John P. Didion1, Kunjie Hua1, Derrick L. Nehrenberg1, Christine L. Powell1, Jill
Steigerwalt5, Yuying Xie1, Samir N.P. Kelada6, Francis S. Collins6, Ivana V.
Yang7, David A. Schwartz7, Lisa A. Branstetter8, Elissa J. Chesler2, Darla R.
Miller1, Jason Spence1, Eric Yi Liu9, Leonard McMillan9, Abhishek Sarkar9,
Jeremy Wang9, Wei Wang9, Qi Zhang9, Karl W. Broman10, Ron Korstanje2,
Caroline Durrant11, Richard Mott11, Fuad A. Iraqi12, Daniel Pomp1,*, David
Threadgill5,*, Fernando Pardo-Manuel de Villena1,* and Gary A. Churchill2,*
1
Department of Genetics, University of North-Carolina-Chapel Hill, Chapel
Hill, North Carolina, USA
2
The Jackson Laboratory, Bar Harbor, Maine, USA
3
Department of Epidemiology, University of North Carolina–Chapel Hill,
Chapel Hill, North Carolina, USA
4
Department of Microbiology and Immunology, University of North Carolina–
Chapel Hill, Chapel Hill, North Carolina, USA
5
Department of Genetics, North Carolina State University, Raleigh, North
Carolina, USA
6
National Human Genome Research Institute, National Institutes of Health,
Bethesda, MD, US
7
National Jewish Health, Denver, CO, USA
8
Oak Ridge National Laboratory, Oak Ridge, TN, USA
9
Department of Computer Science, University of North Carolina–Chapel Hill,
Chapel Hill, North Carolina, USA
10
Department of Biostatistics and Medical Informatics, University of WisconsinMadison, Madison, Wisconsin, USA
11
Welcome Trust Centre for Human Genetics, Oxford University, Oxford, UK
12
Department of Clinical Microbiology and Immunology, Tel Aviv University,
Tel Aviv, Israel
The Collaborative Cross (CC) is an eight-way mouse recombinant inbred strain
panel that is being developed as a resource for mammalian system genetics.
Here we describe an experiment using partially inbred CC lines to evaluate the
genetic structure, mapping power and resolution of the CC. Genome-wide
analysis of high density SNP data in the incipient strains reveals high genetic
diversity, balanced allele frequencies, and dense, evenly distributed
recombination sites – all ideal qualities for a genetic mapping and systems
genetics resource. We mapped white head spotting, a discrete coat color trait;
63
body weight, a highly polygenic complex trait; and more than 11,000 liver gene
expression traits. We demonstrate that analysis based on inferred haplotypes in
the eight-way cross population improves power and provides additional
information about candidate genes not available from standard crosses or strain
surveys. We were able to identify Kitl as a candidate gene for white head
spotting in the WSB/EiJ strain and Asph as a novel candidate gene for body
weight. Genetic mapping of gene expression data demonstrates that resolution
in this experiment is on the order of 1Mb, and this will improve in the finished
CC panel. The number of eQTL discovered here exceeds all previous efforts at
eQTL mapping in mouse strain panels and crosses. The notable absence of
trans- eQTL clustering suggests that multiple functional genetic variants are
evenly distributed throughout the genome of CC mice. We have demonstrated
that the unprecedented genetic diversity of the CC, which derives from random
mixing of an estimated 45 million SNPs, results in high phenotypic diversity and
enhances our ability to map the causative loci underlying complex diseaserelated traits
64
ABSTRACT 28
REALISING THE POTENTIAL OF MOUSE DISEASE MODELS –
FROM PATHWAY TO THERAPY
Steve D.M. Brown
MRC Mammalian Genetics Unit, MRC Harwell, Harwell Science and
Innovation Campus, OX11 0RD, UK
Classical genetic approaches utilising random mutagenesis coupled with focused
phenotyping continues to remain an important tool for discovering novelty in
biological systems. Moreover, the development of a comprehensive mutant
resource for every gene in the mouse genome, followed by large-scale
phenotyping, is also set to transform the available models for dissecting disease
states. The discovery of novelty is the key basis for a cycle of discovery
comprising genetics, molecular, cellular and physiological studies all of which
provide a basis for developing new therapeutic approaches. These paradigms
provide a powerful platform for elucidating the genetic pathways underlying
disease and bringing new insights to the therapeutic strategies that might be
investigated. This is no more important for common diseases for which there is
little genetic understanding and for which current treatments are ineffective. One
such disease is recurrent (ROM) and chronic (COME) forms of otitis media.
ROM and COME are known to have a strong genetic component, but nothing is
known of the underlying genes involved in the human population. Otitis media
with effusion (OME) is the most common cause of hearing impairment in
children in the Western world, potentially causing language delays, learning and
behavioural disruption. The high prevalence of the disease, coupled with its
recurrent and chronic nature, accounts for the large number of ventilation tubes
inserted into the tympanic membrane of affected children. Placement of
ventilation tubes is the most common operation in the UK however the
mechanism by which they work remains uncertain. We have identified two
novel dominant mutants, Jeff and Junbo, which develop a conductive deafness
due to a chronic suppurative OM. The Jeff mutant carries a mutation in an F-box
gene, Fbxo11 (Hum. Mol. Genet. 15, 1-7 2006). Junbo carries a mutation in the
Evi1 transcription factor (PLoS Genetics 2: e149 2006). Evi1 represses the TGFb signalling pathway by the binding of Smad3. Jeff homozygotes die shortly
after birth displaying developmental abnormalities including cleft palate, eyes
open at birth and impaired lung function. TGF-b signalling is involved in all
these epithelial developmental processes and we find that pSmad2 is
significantly upregulated in epithelia of Jeff homozygotes. Mice heterozygous
for both Jeff and Smad2 mutations recapitulate the Jeff homozygous phenotype.
Fbxo11 is known to neddylate p53, a co-factor of pSmad2, and we found that
p53 levels are substantially reduced in Jeff suggesting that Fbxo11 plays a role
in stabilizing p53. Our findings support a model whereby Fbxo11, via
65
stabilization of p53, is required to limit the accumulation of pSmad2 in the
nucleus of epithelial cells. Hypoxia is a common feature of inflamed
microenvironments and the impact of hypoxic mechanisms in OM has received
little attention. Responses to hypoxia are mediated via Hypoxia Inducible Factor
(HIF) and there is considerable cross-talk between TGF-b and HIF-1a pathways
that mediate hypoxic responses. We surmise that dysregulation of TGF-b
signalling in the Jeff and Junbo mutations is having an affect on the response of
the middle ear, including neutrophil and macrophage function, to the hypoxic
environment, leading to the chronic unresolved inflammatory condition. The
role of chronic inflammatory hypoxia and hypoxia-inducible factor (HIF)
mediated responses were investigated in Junbo and Jeff mouse models. Mice
labeled in vivo with Pimonidazole showed cellular hypoxia in middle ear
mucosa and inflammatory cells in the middle ear lumen and there was
upregulation of Il-1b and Tnf-a that modulate HIF. Hif-1a gene expression was
elevated in ear fluid white blood cells and there was upregulation of Vegf
pathway genes including Vegfa gene and protein expression. VEGFR signaling
inhibitors PTK787/ZK 222584, SU-11248 and BAY 43-9006 reduced hearing
loss and modulated inflammatory changes in middle ear mucosa. The
effectiveness of VEGFR signaling inhibitors implies HIF mediated VEGF plays
a pivotal role in otitis media pathogenesis and targeting molecules in HIF-VEGF
signaling pathways has therapeutic potential in the treatment of chronic otitis
media.
66
ABSTRACT 29
IMPORTANCE OF CADM1 AND CELL ADHESION IN DEPRESSIVE
BEHAVIOR
C Santos1, B Miller2, M Pletcher3, A Su4, L Tarantino5, and Tim Wiltshire1
1
University of North Carolina at Chapel Hill, Division of Pharmacotherapy and
Experimental Therapeutics, NC, USA
2
The Scripps Research Institute Jupiter, Florida, USA
3
Pfizer Incorporated Groton, Connecticut, USA
4
Genomics Institute of Novartis Research Foundation at La Jolla, California, USA
5
University of North Carolina at Chapel Hill, Department of Psychiatry, NC, USA
Despite evidence for genetic vulnerability to depressive behavior, genes that can
predict risk or response to anti-depressants have not been identified. Factors
including disease heterogeneity, environmental effects, and complex neurobiological
processes complicate studies in mood disorder. In order to elucidate genetic
mechanisms underlying depression, we have performed a series of experiments
using a panel of genetically diverse mouse inbred strains. We collected genome-wide
gene expression data as well as levels of forty biochemical molecules including
neurotransmitters, neurotrophic factors, neuropeptides, and neuroamine regulators
and receptors under control, fluoxetine, and treatment-naïve conditions from brains
of inbred mice. Furthermore, we have recorded seven baseline anxiety and
depressive behaviors as well as depressive-like responses after administration of
fluoxetine or water across 30-35 mouse inbred strains. Since mice within a strain are
isogenic, inter-strain genotypic and phenotypic differences are analyzed using
haplotype-association mapping algorithm to identify genomic regions associated
with the phenotypes of interest known as quantitative trait loci (QTLs). Candidate
regions were then prioritized based on convergence of evidence from gene
expression, behavior, and biochemical data. A biochemical QTL on Chromosome 9
overlaps with a depressive QTL (which suggests that genes within this region are
associated with behavior as well as biological changes associated with differential
levels of glutamate decarboxylase 67 (GAD67, official symbol GAD1). There are
two genes within this region including cell adhesion molecule 1 (Cadm1) and
2900052N01Rik. Analyses reveal moderate correlation between expression of
Cadm1 and anxiety-like and depressive-like behaviors in addition to levels of several
biochemical molecules. Furthermore, genes within candidate haplotype-depressive
QTL peaks (-logP >3.5) were found to be enriched for genes involved in cell
adhesion. Our findings suggest Cadm1 and other cell adhesion molecules may
mediate molecular and cellular processes that can result in depressive behavior.
Studies are underway to elucidate how cellular adhesive properties modulate
biological changes that lead to depression.
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ABSTRACT 30
AN ENU SENSITIZATION SCREEN TO DETERMINE
PHYSIOLOGICAL FUNCTION OF α- SYNUCLEIN
Deborah E Cabin, M Casey, and D Zou
McLaughlin Research Institute, Great Falls, MT USA
THE
α−synuclein is a small presynaptic and nuclear neuronal protein that is linked to
Parkinson's disease (PD) by its presence in Lewy bodies, the intracellular
inclusions characteristic of sporadic PD, and by mutations responsible for rare
familial forms of that disease. The physiological function of α-synuclein is not
well understood, though several lines of mice that lack the protein have been
studied. Whether or not α-synuclein's normal function is a factor in PD cannot
be assessed until that normal function is better defined. We have taken a
genetic approach to determine α-synuclein's function, a sensitized ENU
mutagenesis screen using mice null for the protein. One hundred twenty
pedigrees have been screened for neurological phenodeviants; 45 lines have
produced non-sensitized phenotypes, 18 lines that transmit phenotypes are being
tested for sensitization, and one confirmed sensitized mutation has been
identified.
Among the neurological phenotypes still being tested for
sensitization are 2 circling mutants, hyperactivity with odd gait, shaky and poor
grip, slow righting and poor grip, lack of exploration, and no response to a
clickbox. The confirmed sensitized mutation is in Atp7a the X-linked transGolgi copper transporter mutated in human Menkes disease. While all males
carrying the Atp7a I610S mutation die regardless of α-synuclein status, Atp7a
mutant females that lack α-synuclein die prior to 35 days at a significantly
higher rate than Atp7a mutant females that express α-synuclein (p= 0.00069,
Χ2). While Atp7a mutations affect many systems in the body, the protective
effect of α-synuclein must be neuronal based on its expression pattern. Menkes
disease has a severe neurological component that can progress to decerebration.
In the absence of α-synuclein, brain steady-state levels of both wild type and
mutant ATP7A protein are highly variable compared to ATP7A levels in mice
that express α-synuclein. The absence of α-synuclein also appears to cause
mislocalization of mutant ATP7A in brain; mislocalization might further impair
the function of a hypomorphic protein. Thus α−synuclein appears to have a
neuroprotective function; in Parkinson's disease, sequestration of the protein in
Lewy bodies may prevent it from performing that function, which may be
particularly important under stress conditions.
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ABSTRACT 31
GENOMIC RESOURCES IN PEROMYSCUS - MAPPING THE
AUDIOGENIC SEIZURE SENSITIVITY LOCUS
Gabor Szalai, Janet Crossland, Jay Coleman, and Michael Felder
Peromyscus Genetic Stock Center, University of South Carolina, Columbia,
SC,USA
A Mice of the genus Peromyscus are among the most abundant mammals in
North America. They range from Alaska to Central America and occur in many
natural habitats. Though superficially resembling laboratory mice (Mus
musculus) and rats (Rattus norvegicus), deer mice are not closely related to
either of these species. One of the major advantages of research with
Peromyscus is their ready adaptability to colony conditions. Research on this
genus has been widespread across so many disciplines that the genus has aptly
been referred to as “The Drosophila of North American Mammalogy”. In order
to make Peromyscus as a viable alternative to Mus, we have been working with
collaborators on developing comparable genomic resources such as an
intermediate resolution genetic map (500 markers to date), synteny map, EST
libraries (50 000 EST clones), BAC libraries and whole genome sequences (to
be completed in December, 2010). These resources are used to map the genetic
locus controlling the audiogenic seizure sensitivity in deer mice. Epilepsy is a
debilitating disease that can arise from either acquired brain lesions or from an
inherited susceptibility to cortical hyperexcitability. At least 40-50% of
epilepsies have a presumed genetic basis. Although few human epilepsy
syndromes are inherited in a simple Mendelian manner, single gene animal
models offer valuable opportunities to isolate gene mutations, to identify
underlying molecular mechanisms and to explore strategies for therapy. In 1935
a spontaneous recessive mutation appeared among laboratory stocks of
Peromyscus maniculatus artemisiae, which has been maintained as a separate
stock since. Using homozygousity mapping analyzing a panel 250 F2 and 100
N2 animals we have identified 4 candidate markers. A comparative cytogenetic
map between Peromyscus maniculatus and Mus musculus reveal that the M.
musculus homologs of the candidate markers on P.m. chromosome 1 and
chromosome 23 would be on M.m. chromosomes 7 and 5, respectively. The two
mapped mouse audiogenic seizure susceptibility genes, mass1 and jams1 are
localized on M. m. chromosomes 13 and 10, respectively. Taken together, our
preliminary data suggests that the genetic mutation underlying seizure
sensitivity in deer mice resides in a novel gene and is not a homolog of a
previously identified susceptibility locus.
69
ABSTRACT 32
MUTATIONS IN THE PDZ DOMAIN CONTAINING PROTEIN GIPC3
CAUSE PROGRESSIVE SENSORINEURAL DEGENERATION (AHL5
AND JAMS1) IN MICE AND RECESSIVE HEARING IMPAIRMENT IN
HUMANS (DFNB95)
Nikoletta Charizopoulou1, Andrea Lelli2, Margit Schraders3,4, Kausik Ray5,
Ronald J.C. Admiraal4, Harold R. Neely1, Joseph R. Latoche1, John K.
Northup5, Hannie Kremer3,4,6, Jeffrey R. Holt2, and Konrad Noben-Trauth1
1
Section on Neurogenetics, 5Section on Signal Transduction, Laboratory of
Molecular Biology, National Institute on Deafness and Other Communication
Disorders, National Institutes of Health, Rockville, MD, USA
2
Department of Neuroscience and Otolaryngology, University of Virginia
School of Medicine, Charlottesville, Virginia, USA
3
Department of Otorhinolaryngology, 6Department of Human Genetics,
4
Nijmegen Centre of Molecular Life Sciences and Donders Institute for Brain,
Cognition and Behaviour, Radboud University Nijmegen Medical Centre,
Nijmegen, The Netherlands
Progressive sensorineural hearing loss affects the quality of life and
communication of millions of people but the underlying molecular mechanisms
remain elusive. Recently, the age-related hearing loss 5 (Ahl5) and juvenile
audiogenic monogenic seizure 1 (jams1) loci were shown to underlie
progressive hearing loss and audiogenic seizures in Black Swiss mice,
respectively. Here, we identify Ahl5 and jams1 as a 343G>A transition in Gipc3
changing the conserved Gly115 to Arg in its PDZ domain and causing a
significant reduction in protein levels. GIPC3 localizes to inner ear sensory hair
cells and spiral ganglia and we show that the mutation disrupts the structure of
the stereocilia hair bundle affecting mechano-transduction currents and longterm survival of spiral neurons. We demonstrate an adverse effect of the
Gipc3343A allele on wave I amplitudes of afferent neurons, which we correlate
with susceptibility and resistance of audiogenic seizures. A Gipc3 transgene
rescues both hearing loss and audiogenic seizures. Lastly, we identify a
truncating mutation in human GIPC3 in a family segregating autosomal
recessive hearing loss DFNB95. Our study reveals a novel and pivotal role of
GIPC3 for the function of the stereocilia hair bundle and for synaptic
transmission.
70
ABSTRACT 33
A MUTATION IN THE GENE ENCODING MITOCHONDRIAL MG2+
CHANNEL MRS2 RESULTS IN DEMYELINATION IN RATS
Takashi Kuramoto1, Mitsuru Kuwamura2, Satoko Tokuda1,2, Takeshi Izawa2,
Yoshifumi Nakane1, Kazuhiro Kitada1,3, Masaharu Akao4, Jean-Louis Guénet5,
and Tadao Serikawa1
1
Institute of Laboratory Animals, Graduate School of Medicine, Kyoto
University, Japan
2
Laboratory of Veterinary Pathology, Osaka Prefecture University, Japan
3
Laboratory of Mammalian Genetics, Genome Dynamics Research Center,
Graduate School of Science, Hokkaido University, Japan
4
Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto
University, Japan
5
Département de Biologie du Développement, Institut Pasteur, France
The rat demyelination (dmy) mutation serves as a unique model system to
investigate the maintenance of myelin, because it provokes severe myelin
breakdown in the central nervous system (CNS) after normal postnatal
completion of myelination. Here, we identified the dmy mutation and
characterized dmy/dmy rats to clarify the pathomechanisms underlying
demyelination. By positional cloning, we found that a G-to-A transition, 177 bp
downstream of exon 3 of the Mrs2 gene, generated a novel splice acceptor site
which resulted in functional inactivation of the mutant allele. Transgenic rescue
with wild-type Mrs2-cDNA validated our finding. Mrs2 encodes an essential
component of the major Mg2+ influx system in mitochondria of yeast as well as
human cells. We found an increased number of mitochondria in the swollen
cytoplasm of oligodendrocytes and elevated serum lactic acid concentration in
mutant rats, indicating that mitochondria were indeed functionally defective.
MRS2-GFP recombinant BAC transgenic rats showed that MRS2 was
dominantly expressed in neurons rather than oligodendrocytes, and
ultrastructurally observed in the inner membrane of mitochondria. These
findings clearly demonstrate that dmy is a loss-of-function mutation of Mrs2 and
suggest that dmy/dmy rats suffer from mitochondrial disease. Our findings imply
that the maintenance and turnover of myelin are genetically independent from its
initial production, and Mg2+ homeostasis in CNS mitochondria is essential for
the maintenance of myelin.
71
ABSTRACT 34
ANNOTATION OF LONG NON-CODING RNA TRANSCRIPTS
Mark Thomas and Jennifer Harrow
HAVANA Group, Wellcome Trust Sanger Institute, Hinxton, Cambridge, United
Kingdom
With the publication of the human genome 10 years ago, came the realisation
that developmental complexity is not related to gene number. Instead, it would
seem that the increased complexity of higher organisms is achieved through
greater regulation of more diverse transcripts. Recent studies would suggest that
a significant proportion of our genome is transcribed, however only 1-2% of the
genome is associated with protein coding loci. A large number of transcripts are
therefore non-coding with no known function and are the subject of much debate
as to whether they are indeed functional or represent transcriptional noise. It is
the remit of the HAVANA group at the Wellcome Trust Sanger Institute to
annotate all transcriptional elements in the human and mouse genomes. As part
of this ongoing effort and in collaboration with the Mattick laboratory and
HGNC, we are proposing a nomenclature system for long non-coding RNA
transcripts (lncRNAs). In the absence of any functional characterisation data for
most transcripts, the proposed schema will ascribe a name for each transcript
based on its genomic context relative to the nearest protein coding locus. We are
also constantly updating our methods to make the most of recent advances in
sequencing technology (eg. RNAseq) and structure prediction methods (eg.
lincRNAs). This has led to the introduction of new non-coding transcript
biotypes, such as antisense, lincRNA, ncRNA-hostgene and 3’overlapping
ncRNA to further define this rapidly emerging class of genes. Furthermore,
analysis of lincRNAs combined with existing data from the Rfam, miRBase and
piRNABank databases have identified potentially new hostgenes for these small
ncRNAs.
72
ABSTRACT 35
THE JAX CRE REPOSITORY: IMPROVING THE UTILITY OF CRE
DRIVER STRAINS
Stephen A. Murray, Caleb Heffner, Michael Sasner, Cathleen Lutz, Brandon
Grossman, Stephen Rockwood, Yashoda Sharma, and Leah Rae Donahue
Capitalizing on mouse gene targeting projects of the International Knockout
Mouse Consortium (IKMC) will require that a large, diverse set of wellcharacterized Cre driver lines. To fill this need, The Jackson Laboratory (JAX)
has committed to increasing the number of Cre lines available to the scientific
community. The JAX Cre Repository currently houses and distributes the single
largest collection of Cre driver strains totaling more than 200 lines, including
156 that are currently distributed as live colonies. The JAX Cre Repository has
embarked on an ambitious project to add value to these strains by
comprehensively characterizing Repository Cre lines. Despite the best efforts of
those developing new Cre lines, the fidelity of Cre activity is not always ideal.
Many difficulties have been reported in various Cre lines, including mosaic or
incomplete deletion in a target tissue/cell type, inconsistent activity, expression
in non-target tissues, and/or Cre-related toxicity. In many cases, however, this
data is not reported or available to the potential user. We have embarked on an
ambitious project to add value to these strains by comprehensively
characterizing all of the Cre lines distributed. We have developed a
comprehensive pipeline for the characterization of Cre driver strains using a
LacZ reporter strain in a wide range of tissues and at multiple time points,
including both target and non-target tissues. In addition, we are examining
possible instances of Cre-induced toxicity, effect of genetic background on
excision fidelity, and functional differences that result from maternal versus
paternal inheritance of the Cre allele. Thus far we have completed the
characterization of 26 strains, and an additional 37 are underway. Our results
indicate the vast majority of Cre driver strains exhibit unexpected recombinase
activity in a number of tissue types, highlighting the need for extended analysis.
We have standardized our data annotation scheme to include 11 broad organ
systems, 30 individual organs/structures and 89 substructures, all of which are
consistent with the mouse Anatomical Dictionary. Slide-scanned images and
associated annotations are published on a dedicated website and submitted to
Creportal.org. This information will allow users to make informed judgments
about the suitability of a particular line for their experiments, and enhance the
power of large-scale mouse gene targeting projects.
73
ABSTRACT 36
MOUSE GENOMES PROJECT: MAP OF SNPS AND SHORT INDELs
Petr Danecek, Gan Xiangchao2, Thomas M Keane1, James Stalker1, Binnaz
Yalcin2, Martin Goodson2, Sendu Balasubramaniam1, Kim Wong1, Guy Slater1,
Andreas Heger2, Eleazar Eskin3, Nick Furlotte3, Chris Ponting, Jonathan Flint2,
and David J Adams1
1
Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire, UK
2
Wellcome Trust Centre for Human Genetics, Oxford, UK
3
University of California, Los Angeles, USA
The Mouse Genomes Project has sequenced the genomes of 17 inbred mouse
strains to between 20-35x coverage, using paired-end Illumina sequencing. One
of the major goals of this project is to generate a comprehensive catalogue of all
SNPs across these strains. Our strategy for calling SNPs was to employ several
next-generation sequencing SNP calling programs and then merge the calls to
produce the final callset. The final set consists of more than 65 millions of
SNPs across the strains. We find extremely high concordance with the Perlegen
SNP set and using 10Mbp of manually finished sequence for NOD/ShiLtJ find a
false positive rate to be 1.4% and false negative rate of 1.7% thus indicating the
high quality of our calls. The final call set consists of 24 thousand coding SNPs
of which 700 were found to be potentially truncating coding mutations. The wild
derived strains such as CAST/EiJ, PWK/PhJ, Spretus/EiJ show an order of
magnitude more variation than the classical strains. From the sequenced based
SNP calls, we have also created an imputed set of SNPs across a further 94
strains. The full set of SNPs has been uploaded to dbSNP and can be queried on
our website which also gives the consequences per SNP, tables of the SNP
patterns across the 17 strains, and enables users to visually inspect the raw
sequence data surrounding individual SNPs.
http://www.sanger.ac.uk/mousegenomes
74
ABSTRACT 37
USE OF PIGGYBAC-MEDIATED TRANSIENT TRANSGENIC RNAI
EXPRESSION FOR RAPID CHARACTERIZATION OF GENE
FUNCTION DURING EMBRYONIC DEVELOPMENT
David R. Beier1, Yuko Fujiwara2, Shannon W. Davis3, Haiyan Qiu1, Thomas L.
Saunders3, Stuart Orkin2, Sally A. Camper3, and Bryan C. Bjork1
1
Genetics Division, Brigham & Women’s Hospital, Harvard Medical School,
Boston, MA, USA
2
Division of Hematology and Oncology, Children’s Hospital, Harvard Medical
School/Howard Hughes Medical Institute, Boston, MA, USA
3
Departments of Human Genetics and Internal Medicine, University of
Michigan, Ann Arbor, MI, USA
The production of targeted mutations in mice remains the gold standard for the
analysis of loss-of-function of specific genes in mammals. However, even with
the emergence of large-scale knockout mouse resources, generation of such
mutants using embryonic stem cells may still require substantial time and
resources. In particular, this approach is difficult to pursue for high throughput
applications. For instance, linkage and association studies for mutations or
strain-specific traits may yield genomic intervals of only moderate resolution,
containing a large number of positional candidate genes. Similarly, microarray
analyses typically result in lists of differentially expressed genes, with little
indication regarding which ones may be key regulators. An efficient
methodology to rapidly screen genes in vivo would enhance the functional
analysis of outputs from high throughput screening. RNA interference (RNAi)
is a powerful strategy for studying the phenotypic consequences of reduced gene
expression. To develop a method for the rapid characterization of the
developmental consequences of gene dysregulation, we tested the use of RNAi
for “transient transgenic” knockdown of mRNA in mouse embryos. These
methods included lentiviral infection as well as transposition using the Sleeping
Beauty (SB) and PiggyBac (PB) transposable element systems. Of the three
methodologies tested, the PB transposon system produced high numbers of
transgenic embryos with the expected phenotype, demonstrating its utility as a
screening method. This approach can be useful for phenotypic validation of
putative mutant loci, as we demonstrate by confirming that knockdown of
Prdm16 phenocopies the ENU-induced cleft palate mutant, csp1. This strategy is
attractive as an alternative to gene targeting in embryonic stem cells, as it is
simple and yields phenotypic information in a matter of weeks. Furthermore, we
found the efficiency of PB for transgenesis was routinely over 65%, suggesting
this method has more widespread applications, and we have adapted it for multigene knock-down, for expression of affinity-tagged proteins, and for analysis of
presumptive transcriptional regulatory sequences.
75
ABSTRACT 38
THE SANGER INSTITUTE MOUSE GENETICS PROGRAMME
Ramiro Ramirez-Solis, J White, E Ryder, R Houghton, and J Bottomley
Mouse Genetics Programme, Wellcome Trust Sanger Institute, Hinxton, UK
The Sanger Institute Mouse Genetics Programme (MGP) capitalizes on the
mutant ES cell resources generated by the EUCOMM and KOMP projects by
generating and phenotyping mutant mouse lines at a large scale. The program
makes the mutant strains and the phenotypic data, available to the scientific
community to galvanize deeper analysis aimed at uncovering the molecular
mechanisms involved in the phenotypic alterations resulting from the mutant
alleles. The MGP has produced over 400 mutant strains, of which more than 200
hundred have finished the phenotypic screen. The phenotypic data can be
obtained
by
visiting
the
Sanger
Mouse
Portal
(http://www.sanger.ac.uk/mouseportal/). The website offers the opportunity to
download a weekly updated summary heat map that includes all the strains
being examined. Scientists are also encouraged to sign up for a phenotypic alert
email list to receive early warnings on interesting phenotypes. When two (2)
heterozygote mice are genotyped for a particular allele, the strain is advertised to
the community at the International Knockout Mouse Consortium (IKMC)
website (http://www.knockoutmouse.org/about). Although the specific mice to
be distributed may not be available immediately, the MGP seeks to receive
notifications of interest, and while the mice are on the shelf, a reasonable effort
is made to distribute them to interested parties with minimum delay. Although a
majority of strains is currently selected because of prior interest on the gene, we
expect that increasingly strains will be selected because of the phenotypic data
generated from the MGP. The selection of phenotypic tests included in the
program is strongly influenced by an active interaction with experts from the
community, and is aimed at exploring a biological space of medical relevance.
We will present an update on the program activities and highlight some
interesting novel phenotypic findings.
76
ABSTRACT 39
NEXT LEVEL SYSTEMIC PHENOTYPING OF MICE
Martin Hrabé de Angelis and the German Mouse Clinic consortium
Helmholtz Zentrum München and Technical University Munich; Institute of
Experimental Genetics, Munich, Germany
Next challenges in functional annotation of mammalian genomes are yet of a
much larger scope than previous genomics initiatives. Mouse mutant resources
must be phenotyped systematically (one after the other) and systemically
(assessing all organ systems). In addition, for the next generation of mouse
models the “envirotypes”, that humans are exposed to need to be modeled. We
established the German Mouse Clinic (GMC) as the first mouse phenotyping
platform worldwide with the logistics of systemic, standardized phenotypic
analysis and interpretation, with open access for the scientific community on a
collaborative basis. The German Mouse Clinic is well integrated into
EUMODIC and Infrafrontier, pan European projects for functional annotation of
mouse models. A new worldwide effort - the International Mouse Phenotyping
Consortium – has been established and will move systemic mouse model
phenotyping to a new scale. To explore the complex relationship between
environmental changes and genetic factors, we have been setting up
standardized challenge platforms for mouse phenotyping. By simulating specific
environmental exposures or life styles we mimic envirotypes that have a strong
impact on human health. Five platforms have been implemented in the areas of
nutrition, exercise, air, infection and stress. Goal is to decipher their effects on
disease etiology and progression, uncovering the physiological and molecular
mechanisms of genome-environment interactions. In addition, identification and
non-invasive screening of biomarkers and risk factors for distinct diseases will
be a future goal in the GMC using a new metabolomic platform and a new
breath-gas analysis set up. First results will be presented.
77
ABSTRACT 41
TOOLING UP MGI FOR A DELUGE IN MOUSE PHENOTYPE AND
DISEASE MODEL DATA
Anna Anagnostopoulos, Janan Eppig, and Mouse Genome Informatics Group
Global efforts to functionally annotate the mouse genome through large-scale
mutagenesis and phenotyping consortia have catapulted the size and diversity of
new mouse resources aimed at building predictive models of human disease.
The Mouse Genome Informatics resource (MGI, www.informatics.jax.org) for
the genetics, genomics and biology of the laboratory mouse grants free access to
current integrated biological knowledge spanning from sequence to phenotype
and disease model information to unified data on worldwide mouse resource
holdings via IMSR. MGI curates aberrant mouse phenotypes in the context of
mutations, strain variations, QTLs and complex traits shaped by intricate
epigenome-environment networks. To support the data deluge fueled by ongoing
translational research, MGI has devised automated processes to gradually
incorporate all multipurpose alleles generated by various IKMC partners, and
built a Recombinase Data Portal (www.creportal.org) to provide expression and
specificity data for each cre-containing transgene and knock-in allele, aiming to
integrate phenotype characterization of newly generated mutant mouse lines as
available. MGI users can access mouse phenotypes, alleles and disease models
of preclinical value using a suite of tools, including an enhanced Quick Search
Tool, Mouse GBrowse, web-based vocabulary browsers and data-specific query
forms. Robust search inputs include standardized terms from the Mammalian
Phenotype Ontology, a widely adopted ontological model that enables
phenotype annotations to background-specific allelic genotypes at varying
degrees of granularity. Use of human disease (OMIM) terms serves to harness
associations between phenodeviant mouse features and orthologous human gene
mutations or disease syndromes for which defined mouse genotypes model the
human condition. We will review MGI phenotype viewing options, customized
retrieval of complex phenogenomic datasets and disease model mining from
various perspectives. Supported by NIH grant HG000330
78
ABSTRACT 42
NEW APPROACHES TO CONDITIONALITY
Aris N. Economides, David Frendewey, Peter Yang, David M. Valenzuela,
Andrew J. Murphy, and George D. Yancopoulos
Regeneron Pharmaceuticals, Inc. Tarrytown, NY 10591, USA
Conditional mutagenesis is rapidly becoming the method of choice for the study
of gene function, but the types of alleles that can be engineered are limited both
by vector design and target gene structure. We therefore developed a new
technology — Conditional-by-Inversion (COIN) — that utilizes an optimized
invertible gene trap-like cassette, the COIN module. The COIN module is
placed in the antisense strand of the target gene, where it is effectively inert until
activated by a recombinase-mediated inversion event that flips the COIN
module into the sense strand, thereby disrupting transcription of downstream
exons while simultaneously providing a reporter for tracking the mutation. The
COIN module can either be inserted into one of the target gene’s natural introns
(an intronic COIN) or directly into a coding exon as part of an artificial intron
(an exonic COIN), greatly increasing allele design flexibility over existing
conditional knockout approaches. The unique artificial intron feature enables
the introduction of the COIN module (or other elements) in nearly any location
within the target gene, freeing up design choices and increasing the types of
alleles that can be designed. We have constructed COIN alleles for a large
number of genes and performed detailed phenotypic analysis on ES cells and
mice before and after activation of the COIN modules. Beyond establishing the
robustness, reliability, and broad applicability irrespective of exon-intron
structure of this method, our study uncovered rare cases of post-inversion
hypomorphic alleles, observed only with intronic COINs, that were caused by
‘skipping’ of the inverted COIN module, resulting in expression of a mixture of
both wild type and COIN-encoding mRNA. We showed that re-engineering the
intronic COIN allele as an exonic COIN by placing the COIN module into the
immediate neighboring exon could rectify this problem. These results not only
inform future design decisions, but may explain the hypomorphism occasionally
observed with traditional gene trap alleles, which are by nature intronic. Finally,
the exon-splitting and reporter features of COINs open up new engineering
modalities for the generation of multifunctional alleles that go beyond
conditional-nulls, ranging from simple splitting of exons to introduce novel
intronic elements to complex multifunctional alleles with more than two
functionalities. Examples of these novel allele designs will be presented.
79
ABSTRACT 43
MOUSE METABOLIC PHENOTYPING CENTERS: NATIONAL
CONSORTIUM FOCUSING ON DIABETIC COMPLICATIONS
Renee LeBoeuf
University of Washington, Seattle, WA, USA
The Mouse Metabolic Phenotyping Center consortium (www.mmpc.org) is
funded by the National Institutes of Health and has the mission to advance
research by investigators using mice primarily for diabetes, diabetes
complications and metabolic diseases. There are six science and one
bioinformatics centers located across the United States of America which
provide unique or well standardized phenotyping tests. We provide in depth
consultations for planning experiments and for data analysis and biological
interpretations. We are also compiling a data base for mouse models studied
within our consortium which is open to NIH and industry-contributing
researchers. We support courses including glucose clamping and tracer usage in
metabolic research, and have two grant awarding mechanisms. The Animal
Models of Diabetic Complications Consortium, made up of individual
investigators who obtain special strains from The Jackson Laboratory, is another
group using our services. Examples of data and MMPC usage will be given.
80
ABSTRACT 46
GENOMIC IMPRINTING: INSIGHTS FROM THE GNAS CLUSTER
Jo Peters
MRC Harwell, Mammalian Genetics Unit, Oxfordshire, UK
Genomic imprinting results in gene expression according to parental origin.
Thus imprinted genes are functionally haploid and so genomic imprinting must
confer significant advantage to offset the cost associated with haploidy. Over
one hundred and thirty well established imprinted genes are known in the mouse
and they tend to occur in clusters. The Gnas cluster contains two protein coding
genes with major phenotypic effects. These are Gnas itself that is maternally
expressed in some tissues and Gnasxl that is exclusively paternally expressed.
Gnas determines the stimulatory G-protein subunit Gsα and Gnasxl encodes a
variant G-protein subunit XLΑS. Both GSA and XLΑS are signal transduction
molecules but they have opposite and potentially antagonistic functions in the
regulation of metabolism and behavior, and each has major effects on preweaning viability. We used a gene targeting approach to investigate how the
imprinted expression of Gnas and Gnasxl is regulated. Imprinted expression
within the cluster is under the overall control of an imprinting control region
(ICR). This region is DNA-methylated on the maternal allele but unmethylated
on the paternal allele. The unmethylated ICR contains an active promoter for a
macro noncoding RNA, Nespas, that we have shown is a cis-acting regulator for
the paternal allele. This finding adds to other evidence that noncoding RNAs are
major elements for regulating imprinted gene expression, although the
mechanisms are not well understood. Nespas runs antisense to Nesp, a paternally
repressed protein coding transcript and our results show that Nespas is required
to silence Nesp so that Gnas can be silenced in turn on the paternal allele. In
addition we have shown that Nespas is required for methylation and therefore
inactivation of the Nesp promoter. Furthermore, a low level of Nespas can
downregulate Nesp in cis through chromatin modification at the Nesp promoter
in the absence of DNA methylation. Our results indicate that a complex set of
RNA and epigenetic interactions have evolved to regulate imprinted gene
expression.
81
ABSTRACT 47
THE BATTLE OF THE SIGNALS: NON-CODING RNAS VS.
ENHANCERS IN THE TRANSCRIPTIONAL REGULATION OF THE
KCNQ1 IMPRINTED REGION
Anjali Raval, Lisa Korostowski, and Nora Engel
Fels Institute, Temple University School of Medicine, Philadelphia, PA, USA
Long non-coding RNAs (lncRNAs) are a growing category of RNAs with
regulatory functions, few of which have been functionally characterized. The
Kcnq1 imprinted domain is involved in Beckwith-Wiedemann syndrome. The
highly conserved mouse cluster is regulated by a paternally expressed 90kb
lncRNA, Kcnq1ot1, that silences most of its neighboring genes, acting strictly in
cis. Kcnq1ot1 is transcribed from an intron of Kcnq1 in antisense direction.
Intriguingly, the paternal Kcnq1 is released from silencing in a tissue-specific
manner, suggesting that strong enhancers can override silencing by the ncRNA.
To study the in vivo spatial organization of the domain and provide insight into
the complex expression patterns, we carried out chromatin conformation capture
(3C) assays. Three-dimensional chromatin topology plays a vital role in bringing
wide-spread regulatory elements into close proximity. We anchored the assays
at the Kcnq1 and Kcnq1ot1 promoters and tested embryos and neonatal tissues
of wild-type mice and mice with a truncation at Kcnq1ot1. Results showed that
interaction frequencies are developmentally regulated and exhibit tissuespecificity. In fact, physical contact between the Kcnq1 promoter and specific
DNA sequences allowed us to identify novel enhancers. Furthermore, we found
major differences in interaction profiles between wild-type and mutant mice.
Transcription of the lncRNA physically restricts the regions that the Kcnq1
promoter can interact with in the wild-type mice, whereas mutant mice
producing a severely truncated ncRNA exhibit promiscuous contacts and
deregulated expression of Kcnq1. These results suggest a unique model whereby
the silencing function of Kcnq1ot1 is exerted by restricting the access of
promoters to specific enhancers by modifying the flexibility of the chromatin
fiber. Nevertheless, strong tissue-specific enhancers can override this effect and
establish the three-dimensional contacts required to promote transcription.
82
ABSTRACT 48
AN IDENTITY CRISIS: ABNORMAL MALE GERM CELL
DEVELOPMENT AND THE INITIATION OF TESTICULAR CANCER
Jason Heaney, Jean Kawasoe, Megan Michelson, and Joseph Nadeau
Case Western Reserve University, Department of Genetics, OH, USA
Testicular germ cell tumors (TGCTs) are the most common cancer in young
men. TGCTs result from anomalies in the development of primordial germ
cells, totipotent embryonic stem cells that normally differentiate into mature
gametes. Spontaneous TGCTs occur at an appreciable frequency only in the
129 family of inbred strains. TGCTs in mice initiate around embryonic day
13.5 (E13.5) during the same developmental time-point at which germ cells
commit to meiosis or mitotic arrest (the mitotic:meiotic switch). In females,
embryonic oocytes pluripotent gene expression decreases and Stra8 expression
is induced to initiates entry into meiosis. In males, pluripotent gene expression
decreases, Stra8 is not induced and gonocytes, the embryonic precursors of
adult male germ cells, become quiescent until after birth when they
differentiate to form spermatogonia. Given the overlap in the timing of TGCT
initiation and the decision to enter meiosis or mitotic arrest, we tested whether
a defect in the mitotic:meiotic switch contributes to TGCT development. We
discovered that TGCT susceptible gonocytes prematurely express markers of
adult spermatogonia and embryonic oocytes between embryonic days E13.5
and E16.5. However, the similarities in differentiation stop at a stage
resembling Ngn3-positive, Kit-negative, undifferentiated A spermatogonia. A
sub-population of TGCT susceptible gonocytes express Ccnd2 but not Kit.
Because expression of both genes occurs at and contributes to male germ cell
differentiation to meiotically-commitment, differentiated A spermatogonia, the
ultimate fate of TGCT susceptible gonocytes may be altered from meiotic to
tumorigenic. To test whether prematurely differentiating, TGCT susceptible
gonocytes commit to meiosis, we immunolabeled chromosome spreads for
components of the synaptonemal complex (SCP3 and SCP1). Interestingly,
axial elements form but synapsis does not occur in TGCT susceptible
gonocytes. These results suggest that prophase I of meiosis is initiated but
aborted in TGCT susceptible gonocytes. We recently discovered that
suppression of pluripotent gene expression is delayed in TGCT susceptible
gonocytes. Interestingly, in vitro stimulation of pluripotent gene expression in
undifferentiated A spermatogonia induces the formation of pluripotent stem
cells with teratoma forming capacity. Similarly, TGCT formation in vivo may
result from the establishment of A spermatogonia-like cells with pluripotent
stem cell properties. Ongoing studies are testing the influence of retinoic acid
on TGCT susceptibility and the contributions of Stra8 expression and meiotic
initiation to TGCT formation.
83
ABSTRACT 49
AGE-ASSOCIATED CHANGE OF ENERGY METABOLISM ARE
GENETICALLY DISSECTED BY MOUSE INTER-SUBSPECIFIC
CONSOMIC STRAINS
Toyoyuki Takada1, Akihiko Mita1, Shigeharu Wakana2, Kazuo Moriwaki2,
Hiromichi Yonekawa3, and Toshihiko Shiroishi1
1
National Institute of Genetics, Shizuoka, Japan
2
RIKEN BRC, Ibaraki, Japan
3
Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
Energy metabolism-related traits including fat deposition are governed by
environmental and complex genetic factors, which are also largely influenced by
aging with concomitant change in basal metabolic rate. In this study, we intend
to uncover the genetic determinants that control the age-associated changes of
energy metabolism by functional genomics with mouse inter-subspecific
consomic strains. We have established a full set of consomic (chromosome
substitution) strains, B6-ChrNMSM, with each of C57BL/6 (B6) chromosomes
replaced by its counterpart of MSM/Ms, which was derived from M. m.
molossinus. Using the panel of consomic strains, we conducted systematic
phenotype screening of body growth and physiological traits by 10 weeks of
age, and successfully detected several hundred of energy metabolism-related
QTLs (http://molossinus.lab.nig.ac.jp/). Currently, we extend the period of
observation of the same traits to 25 weeks of age. Comparing phenotype of each
strain at the two different observation periods, we systematically explored
strains that show significant change in measurement values. We found that many
strains drastically changed the values of energy metabolism-related traits when
compared with change of the consomic background strain B6. For examples,
consomic strains of chromosome 11 and 15 showed extremely low rate in
increase of fat deposition. This suggests that although wild-derived MSM/Ms
strain has thrifty-type metabolic pathway as a whole, it also carries genetic
factors leading to age-associated anti-obesity. Thus, this study shows that the
inter-subspecific consomic strains would provide powerful tools to dissect ageassociated change of energy metabolic-related traits.
84
ABSTRACT 50
GENETIC REGULATION OF AGING RELATED PHENOTYPES AND
LIFESPAN IN MICE
Rong Yuan1, SW Tsaih1, Q Meng1, K Flurkey1, J Nautiya2, SB Petkova1, MA
Bogue1, KD Mills1, LL Peters1, CJ Bult1, CJ Rosen1, JP Sundberg1, M Parker2,
DE Harrison1, GA Churchill1, and B Paigen1
1
The Jackson Laboratory, Maine, USA
2
Imperial College London, London, UK
To better characterize aging in mice, the Jackson Aging Center carried out a
lifespan study of 31 inbred strains. Clinical assessments were carried out every 6
months, measuring multiple age-related phenotypes including development
traits, organ functions, body composition, hematology, hormonal levels, and
immune system parameters. In a concurrent cross-sectional study of the same 31
strains at 6, 12, and 20 months, more invasive measurements were carried out
followed by necropsy to assess apoptosis, DNA repair, chromosome fragility,
and histopathology. Survival curves varied dramatically among strains. The
median lifespans ranged from 251 to 964 days. By conducting correlation
analyses, we found some phenotypes correlated with lifespan. Particularly,
plasma IGF1 levels showed an inverse correlation with a median lifespan at 6
months (R =-0.33, P = 0.01). This correlation became stronger if the short-lived
strains with a median lifespan < 600 days were removed (R = 0.53, P < 0.01).
IGF1 levels at 6 months also significantly correlated with the ages of vaginal
patency (AVP), a measurement for sexual maturation (R=-0.44, P=0.01). AVP
positively correlated with median lifespan, however it is not significant (P=0.22)
until the short-lived strains were excluded (R=0.38, P<0.05). Haplotype
association mapping identified three loci — Vpq1, 2, 3 — on Chromosomes 4
and 16 that significantly associated with the variation in AVP. At these loci, all
four wild-derived strains — WSB/EiJ, CAST/EiJ, PWD/PhJ, MOLF/EiJ — each
of which represents a different sub-species of the mouse family, have the same
haplotypes, which differ from those of almost all domesticated inbred strains. A
consomic strain, which carries chromosome 16 of PWD/PhJ on the C57BL/6J
background, had significantly delayed AVP. Bioinformatic analysis suggested
that nuclear-receptor-interacting protein 1 (Nrip1) is a candidate gene for Vpq3.
AVP was delayed in the Nrip1 knock out mice compared to controls,
demonstrating that Nrip1 can influence the age of sexual maturation. These
results suggest common genetic mechanisms may exist for regulating IGF1
levels, AVP and lifespan. Using aging related phenotypes as entrées may
facilitate the identification of genes that regulate aging and longevity.
85
ABSTRACT 51
SUBSPECIFIC ORIGIN AND HAPLOTYPE DIVERSITY IN THE
LABORATORY MOUSE
Hyuna Yang1, Jeremy R Wang2, John P Didion3, Ryan J Buus3, Timothy A Bell3,
Catherine E Welsh2, François Bonhomme4, Alex Hon-Tsen Yu5, Michael W
Nachman6, Jaroslav Pialek7, Priscilla Tucker8, Pierre Boursot4, Leonard
McMillan2, Gary A Churchill1, and Fernando Pardo-Manuel de Villena3
1
The Jackson Laboratory, Bar Harbor, ME 2Department of Computer Science; 3,
Center for Genome Science, University of North Carolina Chapel Hill, NC, USA
4
Université Montpellier, Institut des Sciences de l'Evolution, Montpellier,
France
5
Institute of Zoology and Department of Life Science, National Taiwan
University, Taipei Taiwan ROC 10617
6
Department of Ecology and Evolutionary Biology, University of Arizona,
Tucson, AZ, USA
7
Department of Population Biology, Institute of Vertebrate Biology, Studenec,
Czech Republic
8
Department of Ecology and Evolutionary Biology, University of Michigan, Ann
Arbor, MI, USA
In this study we provide the first genome-wide, high-resolution map of the
phylogenetic origin of the genome of the majority of extant laboratory mouse
inbred strains. Our analysis is based on the genotypes of wild caught mice from
three distinct subspecies of Mus musculus. We demonstrate tha classical
laboratory strains are derived from a small pool of fancy mice with limited
haplotype diversity. Their genomes are overwhelmingly M. m. domesticus in
origin and the remainder is mostly of Japanese (M. m. molossinus) origin. We
have generated genome-wide maps of haplotype sharing in classical inbred
strains based on identity by descent from fancy mice and demonstrate that,
despite broad phenoptypic diversity, classical inbred strains have limited and
non-randomly distributed genetic diversity. In contrast, wild-derived laboratory
strains represent a broad sampling of diversity within the species M. musculus.
However, intersubspecific introgression is pervasive in these strains and
contamination by laboratory stocks has played an important role in this process.
The subspecific origin, haplotype diversity and identity by descent map in
laboratory strains can be visualized and searched at www.cgd.jax.org. This
work provides a detailed characterization of the evolutionary origins of this
widely used model for biomedical research.
86
ABSTRACT 53
GENETIC ARCHITECTURE OF HYBRID STERILITY: MATCHING
THE PUZZLE PIECES TOGETHER
Jiri Forejt, Maria Dzur-Gejdosova, Vaclav John, Tanmoy Bhattacharyya, Petr
Simecek, Sona Gregorova, Ondrej Mihola, Petr Flachs, and Zdenek Trachtulec
Institute of Molecular Genetics and Center for Applied Genomics, Academy of
Sciences of the Czech Republic,Videnska, Czech Republic
Hybrid sterility genes are involved in reproductive isolation and consequently in
speciation by restricting gene flow between related taxa. The house mouse Mus
m. domesticus and Mus m. musculus, two subspecies in statu nascendi, represent
unique mammalian models to study speciation because of their recent
evolutionary divergence, and because of the availability of variety of genetic
tools and sequence information. We are using C57BL/6J (B6) inbred strain as a
model of Mus m. domesticus (cca 95% of its genome is of domesticus origin)
and PWD/Ph wild-derived inbred strain of musculus origin. All F1 hybrid males
from crosses of PWD females and B6 males are sterile with no sperm. By
substituting B6.PWD-Chr# consomics for B6 parent in PWD x B6 hybrid cross,
we tested, separately for each chromosome, the necessity of PWD/B6
heterozygosity for male sterility. Using these crosses we found that PWD/PWD
homozygosity of Chr 17 or Chr 19, and B6 form of Chr X rescue hybrid sterility
while PWD/PWD genotype of all other autosomes did not interfere with sterility
of hybrid males. In independent experiments, we mapped hybrid sterility genes
as QTLs using several genetic backcrosses, F2 intercrosses and testcrosses. At
least 3 hybrid sterility loci were identified, in agreement with the consomic
mapping. On Chr 17 we identified Hybrid sterility 1 locus with Prdm9 gene and,
more recently, we localized the Hstx2 gene on Chr X. Genetic mapping of the
gene(s) responsible for hybrid sterility rescue on Chr 19 is in progress. Detailed
analysis of intrameiotic block characteristic for (PWD x B6) hybrid male
sterility revealed failure of meiotic pairing of variable number of autosomes.
The unpaired autosomes, differentially decorated by SYCP1 and SYCP3
antibody, displayed failure of DSB repair at pachynema marked by the
persistence of ATR, RAD51 and γH2AX. The X chromosome showed lack of
di-methylation of histone H3K9, and transcription profiling of prepubertal testes
and FACS- sorted primary spermatocytes indicated improper inactivation of the
X chromosome. Thus the interference with meiotic male sex chromosome
inactivation (MSCI) could represent the major surveillance mechanism of hybrid
sterility that could be under the control of at least three hybrid sterility genes.
We want to propose that the interference with the MSCI is the major molecular
mechanism explaining the Haldane’s rule in gametogenesis of interspecific
hybrids.
87
ABSTRACT 54
MOLECULAR INTERACTIONS OF DEAD END (DND1)
Angabin Matin1, Zhu Rui1, KangLi Luo1, Chitralekha Bhattacharya1, Michelina
Iacovino2, Elisabeth Mahen2, Michael Kyba2 and Sita Aggarwal3
1
University of Texas, MD Anderson Cancer Center, Houston, Texas, USA,
2
University of Minnesota, Minneapolis, USA
3
Pennington Biomedical Research Center, Baton Rouge, Louisiana, USA
Inactivation of the Dead End (Dnd1) gene in mice results in sterility or
development of testicular germ cell tumors in a strain specific manner. Dnd1
encoded protein, DND1, possesses canonical RNA recognition motifs. It has
been shown that DND1 binds to the 3’-untranslated regions (UTRs) of mRNAs
to block microRNA (miRNA) mediated inhibition of translation. As DND1
helps maintain translation from specific mRNAs, it is expected that inactivation
of DND1 will result in loss of cell-type specific protein expression and cell
death. DND1 is expressed in germ cells as well as in embryonic stem (ES) cells.
Early germ cells and ES cells share gene expression patterns, markers and
miRNAs in common. For example, both cell types express markers such as
POU5F1, VASA, NANOG and FRAGILIS. They express similar miRNA
families (example, miR 209-295, miR 302-367 and miR 17-92) and pluripotency
factors such as POU5F1, SOX2 and NANOG. Indeed, there is speculation that
ES cells may be derived from germ cells of the early embryo. Because ES cells
express DND1, it is expected that they also express the mRNAs and miRNAs
whose activity is modulated by DND1. Therefore, we generated a stable,
modified ES cell line that expresses tagged DND1 in a regulatable manner. We
used this line to perform ribonucleo immunoprecipitation (RIP) assays followed
by RT-PCR. We found that the mRNA targets of DND1 in ES cells include
pluripotency factors, apoptotic factors and tumor suppressors. We will present
our RIP results of candidate DND1 mRNA targets from ES cells. Because
protein levels of many factors are precisely regulated in ES cells, our results
suggest that DND1 likely imposes another level of translational regulation of
critical factors in ES cells. Second, we examined the effect of DND1 interaction
with the cytidine deaminase, APOBEC3. We had previously demonstrated that
APOBEC3 interacts with DND1. Using luciferase reporter assays we found that
APOBEC3 can block DND1 activity and restore miRNA mediated translation
repression of p27. Based on our results we hypothesize that the APOBEC3DND1 interaction may be involved in providing specificity of DND1 function.
In summary, our results indicate that DND1 likely imposes another level of
translational regulation to control expression of critical factors in ES cells. In
addition, interaction of DND1 with other proteins such as APOBEC3 may
provide further specificity of DND1 function. These studies are beginning to
shed light on the role of DND1 in cell death and transformation.
88
ABSTRACT 55
GENETIC
DISSECTION
OF
METATASIS
SUSCEPTIBILITY
CONVERGES ON A COMMON MECHANISM
J Alsarraj, S Winter, N Goldberger, K Mattaini, M William, L Lukes, R Walker,
and Kent W Hunter
Laboratory of Cancer Biology and Genetics, National Cancer Institute, NIH,
Bethesda, MD, USA
Metastasis, or dissemination and growth of tumors at secondary sites, remains a
major unsolved problem that accounts for the majority of cancer related
mortality. The metastatic process is a complex cascade of events that includes
escape from the primary tumor, invasion through surrounding tissue, penetration
of the vasculature, resistance to anchor-independence induced apoptosis, arrest
in the secondary site, exiting from the vasculature and finally growth in a novel
microenvironment. Acquisition of these abilities has traditionally been thought
to be due to the accumulation of somatic mutations in a small subset of cells
during tumor evolution.
More recently however, our laboratory has
demonstrated that there is a significant inherited susceptibility for breast cancer
metastasis in both mouse and human populations. Using a quantitative trait
strategy we have identified and validated a number of metastasis susceptibility
genes, including the genes Sipa1, Brd4, and Rrp1b. Applications of additional
genome-wide analytical tools have revealed additional candidate metastasis
susceptibility genes from our genetic mapping crosses. Unexpectedly, many of
the genes identified appear to encode proteins that operate within the same
transcriptional control and chromatin modification pathway, physically interact,
and oppose each others function. These data suggest that inherited metastatic
susceptibility is due to modification of a complex mechanism regulating basal
transcriptional control of breast epithelial cells.
89
ABSTRACT 56
Icst IS A DOMINANT NEGATIVE MUTATION OF Lmx1b
Sally H. Cross1, Lisa Mckie1, Margaret Keighren1, Dan Macalinao2, Alison L.
Kearney2, Simon W. John2,3, and Ian J. Jackson1
1
MRC Human Genetics Unit, Edinburgh, UK
2
The Jackson Laboratory, Bar Harbor, ME USA
3
Howard Hughes Medical Institute, The Jackson Laboratory, Bar Harbor, ME
USA
The ENU-induced mutation iris-corneal strands (Icst) causes raised intraocular
pressure, bulging eyes and anterior segment scarring when heterozygous and is
recessive lethal. Homozygotes have limb and skull abnormalities and absent
cerebellum. Icst is a missense mutation of the LIM-homeodomain transcription
factor Lmx1b that prevents the binding of the mutant protein to its target DNA
sequence. Mutations in LMX1B underlie the dominant human disorder nailpatella syndrome (NPS). This is a pleiotrophic disorder characterised by nail and
joint dysplasia that is sometimes accompanied by kidney defects and glaucoma.
It is thought to be caused by haploinsufficiency, although there is great
variability in disease aetiology both within and between families. Lmx1b has
been knocked-out in the mouse and although the reported homozygous
phenotype is very similar to that of Icst, heterozygous knock-out mice are
reported to be normal, in contrast to the strongly penetrant eye phenotype of Icst
heterozygotes. Both mutant models are on C57BL/6 suggesting strain
background is unlikely to account for the observed phenotypic differences. We
hypothesised that Icst mutant LMX1B protein has a dominant negative mode of
action and that the relative level of wild-type to mutant protein is crucial. We
used recombineering to introduce the Icst mutation into an Lmx1b-containing
BAC and made transgenic lines with both the wild-type and the mutant BAC.
Both express the Lmx1b transgene at 65-70% the level of the endogenous gene.
One copy of the wild-type BAC rescues the dominant eye phenotype and two
copies rescue the homozygous lethality but not the limb phenotype. The limbs of
transgenic rescued homozygotes are symmetrically ventralised, indicating that
although the absolute level of wild-type LMX1B protein is greater than in Icst
heterozygotes, the excess mutant protein prevents normal development.
Furthermore, when we introduce the mutant Lmx1b transgene into Icst
heterozygotes we see embryonic lethality, demonstrating that a normally
sufficient, heterozygous, wild-type gene is insufficient when there is an
increased dose of mutant protein. It has previously been suggested in humans
that mutant LMX1B alleles do not influence the wild-type allele. However, we
show that the gene can have dominant negative mutant forms, and some cases of
NPS could be due this mechanism.
90
ABSTRACT 57
ELUCIDATING THE ROLE OF NSDHL AND CHOLESTEROL
METABOLISM IN CNS DEVELOPMENT USING A CONDITIONAL
KNOCKOUT ALLELE
Gail E Herman1, N Bir1, L Binkley1, K McLarren2, C Boerkoel2, and D
Cunningham1
1
The Research Institute at Nationwide Children’s Hospital, Columbus, OH, USA
2
Department of Medical Genetics, University of British Columbia, Vancouver,
BC, USA
NSDHL (NADH steroid dehydrogenase-like) is a 3β-sterol dehydrogenase
involved in the removal of C-4 methyl groups in one of the later steps of
cholesterol biosynthesis. Mutations in the murine gene are associated with the
X-linked, male-lethal mouse mutations bare patches (Bpa) and striated (Str).
Intellectual developmental disabilities (IDD) and developmental CNS
malformations are prominent features of human disorders of cholesterol
synthesis. In addition, abnormalities of cholesterol metabolism have been
implicated in more common CNS disorders ranging from autism to
Alzheimer’s. Recently, two families with X-linked IDD in males have been
found to have hypomorphic human NSDHL mutations. In one family, female
carriers exhibited significant callousness (P=0.002), linking cholesterol and
behavior. To begin to understand the role of cholesterol and its intermediates in
CNS development and behavior, we generated a conditional Nsdhl targeted
allele (Nsdhlflx5). The “floxed” animals are fertile and without any obvious
phenotype. Nsdhlflx5/+ females mated to Sox2-Cre males exhibit an identical
phenotype to that observed in surviving Bpa1H/+ females carrying a null allele.
There was also no detectable NSDHL protein in Sox2-cre deleted E9.5 or
E10.5 male embryos. Nsdhlflx /Nsdhlflx5 females were mated to nestin, GFAP, or
Thy1 cre males that have cre expression in neural precursors starting at E10.5;
radial glia at E13.5; and selected cortical neurons at P15, respectively. No
liveborn deleted NsdhlnesΔ5/Y have been recovered, while Thy1 cre deleted
mice have no overt phenotypes at >100 days of age. Hemizygous GFAP cre
deleted males develop early postnatal ataxia (P10-12), wasting, and cerebellar
degeneration with massive apoptosis and death by P15-P20. While early
cerebellar differentiation appears normal, cells of the external granule layer do
not appear to exit the cell cycle and migrate properly. In addition, although
GFAP cre is not expressed in Purkinje cells, these neurons also die, suggesting
that they require the continued presence of supporting glia or are exposed to
toxic metabolites. The hippocampus similarly exhibits neuronal cell loss
beginning in the late prenatal period. Possible mechanisms, including altered
cell signaling and accumulation of toxic intermediates, will be discussed.
91
ABSTRACT 58
GENETIC, STEM CELL, AND SYSTEMS ANALYSES OF
NEURODEGENERATIVE DISEASES
George A Carlson1, R Bennett1, ME Orr1, I Lee2, H Yoo2, J-H Cho2, D Hwang3,
and LE Hood2
1
McLaughlin Research Institute, Great Falls, Montana, USA
2
Institute for Systems Biology, Seattle, Washington, USA
3
POSTECH, Pohang, Republic of Korea
Prions cause transmissible neurodegenerative diseases and replicate by
conformational conversion of benign forms of prion protein (PrPC) to diseasecausing PrPSc isoforms. Tracking global gene expression changes in the brains
of 8 distinct mouse strain-prion strain combinations that differed widely in
disease incubation time enabled capture of the effects of prion strain, host
genetics, and PrP concentration. A core of 333 differentially expressed genes
(DEGs) appear central to prion pathogenesis. Most of these core DEGs appear
before clinical disease but are related to pathological processes. Core DEGs
also change in PrP-overexpressing transgenic mice with very short incubation
times, but the magnitude of differential expression is smaller and closer in time
to clinical illness than in other mouse strain-prion strain combinations. The most
significant DEGs in these transgenic mice change early and are not seen in the
other combinations; we hypothesize that these DEGs may reflect the earliest
responses to prion infection and that their gene products may be involved in
prion replication. To test this, CNS stem cell-containing neurosphere cultures
were infected with mouse prions. Prior to induction of differentiation, no
obvious pathogenic effects of prion replication on neurospheres were observed;
thus, DEGs found in prion-infected neurospheres likely reflect processes related
to prion replication. This systems approach is enabling identification of
intersecting networks of genes crucial for establishing and maintaining prion
infection and will help define the link between replication and pathogenesis.
Similar approaches are now being applied to a mouse model for frontotemporal
dementia with the ultimate goal of identifying gene and protein expression
signatures that can discriminate among diverse neurodegenerative diseases.
92
ABSTRACT 59
STAGE AND CELL SUBTYPE-SPECIFIC EPIGENETIC REGULATION
OF MAMMARY GLAND DEVELOPMENT
Deanna Acosta1, Melissa J Fazzari1, John M Greally1, and Cristina Montagna1
1
Albert Einstein College of Medicine, Bronx, NY, USA
Postnatal development of the mammary gland is a complex and highly
specialized process characterized by cell proliferation and tissue remodeling.
Many genes involved in mammary gland development have been identified and
their expression extensively investigated at all major developmental stages.
However, the means by which these genes are regulated have not yet been
established. Despite advances in understanding the role of epigenetics in gene
regulation, little is known about the relationship between cell subtype-specific
cytosine methylation and gene expression in the developing mammary gland.
Based on previous reports that cytosine methylation is dynamic during
development and that cell type-specific methylation affects gene expression, we
hypothesize that alterations of DNA methylation levels occur during
postnatal mammary gland development in a stage- and cell subtype-specific
manner. We propose that these changes act as one of the mechanisms
regulating the expression of transcription factors that drive development
and differentiation. Using a known gene in breast development (Serpinb5) we
determined if cytosine methylation changes occur during mouse mammary
gland development and if these changes correlate with expression. Serpinb5 is
regulated by alterations in cytosine methylation in breast cancer cells. Our
preliminary studies suggest that the Serpinb5 promoter region is differentially
methylated through the developmental stages and that its methylation status
correlates with changes in expression. To identify novel candidate loci, we
performed a genome-wide analysis of cytosine methylation levels during
mammary gland development. Our study indicates that conserved, non-coding
regions around the transcription start sites of a subset of genes undergo changes
in methylation during development. Of these, we selected Serpinb5, Gata3,
Tnfsf11 (Rankl) Cebpb, and Cebpd because they have been shown to play
critical roles in mammary gland development whereas Gata2, a novel candidate
gene, is involved in the development of other cell types. To approach the issue
of mammary gland tissue heterogeneity, we sorted luminal and myoepithelial
cells from different developmental stages. With these cell subtypes, we aim to
establish a genome-wide methylation profile and validate the role of cytosine
methylation in the regulation of our candidate genes during mammary gland
development. This study would not only uncover the role of cell subtypespecific cytosine methylation in tissue morphogenesis, but it will help pinpoint
genes that could be altered at early stages of breast tumorigenesis.
93
ABSTRACT 60
TOWARD AN UNIFIED MEASURE OF INTRASPECIFIC SELECTIVE
PRESSURE
Roberto Amato1,2, Gennaro Miele1,2, Michele Pinelli1,3, and Sergio Cocozza1,3
1
Gruppo Interdipartimentale di Bioinformatica e Biologia Computazionale,
Università di Napoli "Federico II" & Università di Salerno, Italy
2
Dipartimento di Scienze Fisiche, Università di Napoli "Federico II" - INFN
Sezione di Napoli, Naples, Italy
3
Dipartimento di Biologia e Patologia Cellulare e Molecolare "L. Califano",
Università di Napoli "Federico II", Naples, Italy
During recent decades the study of human evolution has been of increasing
interest, also due to the large amount of data now available. In the mean time,
new applications of evolutionary biology to medical problems are being
discovered at an accelerating rate. Several estimators for the selective pressure
have been proposed. Being introduced to face different aspects of selective
pressure, each measure has its own pros and cons. We focused on the most
widely used intraspecific estimators of selective pressure. In particular, to cope
with both inter- and intra-group phenomena, we analyzed the fixation index
(FST) and some measures based on the extended haplotype homozygosity
(namely REHH, iHS, XP-EHH). We assessed the performances of each measure
on simulated data produced using Fregene, a tool developed and calibrated to
reproduce, in a biologically sound manner, our evolutionary history, also taking
into account for complex demographic, selection and recombination scenarios.
By using GRID facilities to produce a large enough amount of data, we were
able to exhaustively assess the performances of each estimator with regard to
several biological parameters of the sites under selection (e.g. selection
coefficient, dominance model, age of selection). Our analysis highlighted a
complementary behavior of these estimators suggesting a possible strategy of
merging them in an unified and versatile measure of selective pressure.
94
ABSTRACT 61
A NEW TARGETED MUTATION, CASPA, IN THE GNAS
COMPLEX SHOW S HYPERACTIVITY AND ATAXIA
Simon Ball1, Sally Eaton1, Christine Williamson1, Charlotte Tibbit1,2, and Jo
Peters1
1
MRC Harwell, Mammalian Genetics Unit, Oxon, UK
2
Current address: MRC Functional Genomics Unit, Oxford, UK
Imprinted genes, by definition, are preferentially expressed from either the
maternally or paternally derived allele. They are known to affect growth,
survival and behaviour. We report here a new targeted mutation, Caspa, within
the Gnas imprinted gene complex. On maternal inheritance, Caspa/+ mice
appear chunky have a tail kink or bend and most die before weaning. Notably
they are hyperactive, crawling by the day after birth and running by 7-8 days,
several days before their wild-type sibs. Ataxia has been seen in some several
days after birth, but this is not associated with cerebellar defects. The
hyperactivity is reminiscent of that seen in mice with paternal uniparental partial
disomy for distal chromosome 2 (PatDp.dist2). In both Caspa/+ and
PatDp.dist2, Gnasxl, a paternally expressed transcript in the Gnas complex, is
expressed at twice the normal level. We suggest that over expression of Gnasxl,
that encodes a variant alpha subunit XLαs of the signalling Gs protein, is the
cause of the hyperactivity.
95
ABSTRACT 62
RAT RESOURCE AND RESEARCH CENTER
Beth A. Bauer, EC Bryda, CL Franklin, LK Riley, and JK Critser
Department of Veterinary Pathobiology, Research Animal
Laboratory, University of Missouri, MO, USA
Diagnostic
The Rat Resource and Research Center (RRRC) was established in 2001 with
funding from the National Institutes of Health (NIH) National Center for
Research Resources (NCRR). The goals of the RRRC are to (1) provide the
biomedical community with a repository and distribution center for valuable rat
strains, and (2) to shift the burden for maintaining and distributing unique rat
models from investigators to a National Resource Center. Currently, the RRRC
has more than 160 rat lines received through active recruitment of valuable rat
models and donations from investigators who have created models. Upon
importation into the RRRC, gametes and embryos are cryopreserved to insure
against future loss of the model. Recent additions to the repository include a
collection of ENU and transposon-generated mutants from the Medical College
of Wisconsin. Also, newly developed rat embryonic stem cell lines have been
imported into the RRRC and are available for distribution to the research
community. Models in the RRRC are available for distribution as live animals,
tissues, or cryopreserved gametes and embryos. In addition to repository and
distribution functions, the RRRC also conducts research. To date, research
efforts by the RRRC have significantly advanced cryobiology and assisted
reproductive technologies (ART) for the rat. Due to high success rates with
intracytoplasmic sperm injection (ICSI), the RRRC can use sperm
cryopreservation as a cost effective method for banking large collections of
single gene mutations and ensuring reliable recovery when models are
requested. The RRRC (http://www.rrrrc.us) is a valuable resource to the
community of rat users as it continues to expand the number of accessible rat
models and services available to the biomedical community.
96
ABSTRACT 63
LOCI ON CHROMOSOME 2 ARE ASSOCIATED WITH NAÏVE
AIRWAY HYPERRESPONSIVENESS IN CD-1 OUTBRED MICE
David R Beier1, Andrew Kirby2, Mary Prysak1, Mark Daly2, and Emily Cozzi1
1
Department of Medicine, Division of Genetics, Brigham and Women’s
Hospital, Harvard Medical School, Boston MA, USA
2
Center for Human Genetic Research, Massachusetts General Hospital,
Harvard Medical School, Boston, MA, USA
Asthma is a complex syndrome characterized by episodic and reversible airway
obstruction, airway inflammation, and airway hyperresponsiveness. The genetic
mechanisms predisposing individuals to asthma and in particular airway
hyperresponsiveness (AHR) are largely unknown. Quantitative trait linkage
analyses (QTL) have been performed to identify loci associated with naive AHR
in a limited number of inbred murine populations. The goal of this study was to
uncover loci associated with AHR in a genetically heterogeneous outbred
murine population. Outbred CD1 mice were intercrossed to generate F1 progeny
from four male/female breeding pairs (N=88). All mice were phenotyped by
determining methacholine-induced airway resistance with the invasive flexiVent
system. CD1 F1 mice were genotyped using an Illumina medium density 1440
SNP panel and linkage analysis was carried out using Merlin and GeneHunter
software. The CD1 F1 progeny had a highly variable AHR phenotype (2.2-21.5
cmH2O.s/mL). Linkage analysis of this population was done by inferring
parental haplotypes; this identified loci on chromosome 2 as being significantly
associated with the AHR phenotype in 2 of the 4 families (additive LOD score =
3). Remarkably, this data is consistent with what we have previously discovered
for the A/J and AKR/J inbred hyperresponsive strains. In order to obtain high
resolution of the loci identified on chromosome 2, CD1 F1 mice were genotyped
on the JAX 600K SNP Diversity Array panel. Analysis of this data may
facilitate the identification of specific haplotypes associated with the AHR trait.
In summary, we have found a robust distribution and segregation of AHR as a
trait in outbred CD1 F1 mice, and identified loci on chromosome 2 that are
significantly associated with AHR. Overall, these data highlight that common
AHR loci may exist between hyperresponsive murine strains.
97
ABSTRACT 64
HIGH-RESOLUTION MAP AND CANDIDATE GENE ANALYSIS FOR
ÉBOURIFFÉ (EBO), A HAIR MUTATION ON MOUSE CHROMOSOME
2 ASSOCIATED WITH INFERTILITY
Fernando Benavides1, J Jaubert2, CJ Perez1, J-L Guénet2, J Barrera1, B Cole1,
and CJ Conti1
1
Department of Carcinogenesis, M.D. Anderson Cancer Center, Science ParkResearch Division, Smithville, TX
2
Unité de Génétique Fonctionnelle de la Souris, Institut Pasteur, Paris, France
Forward genetics is a powerful approach to discover genes with pleiotropic
effects in the mouse. We previously described ébouriffé (ebo), a mouse mutation
with a hair phenotype and infertility (male and female). This autosomal
recessive mutation arose spontaneously in a substrain of BALB/c and was
localized on proximal chromosome 2 using an interspecific backcross. Affected
ebo/ebo mice show curly vibrissae and wavy hair from day P7, and males have
defective spermatogenesis with structural abnormalities of the acrosome that
lead to abnormal head shapes and isolated flagellum (Biology of Reproduction
55: 355-363, 1996). New mapping crosses allowed us to narrow down the
segment of Chromosome 2 containing ebo to a ~1 Mb interval, between markers
D2Mit153 and D2Mit64 (29.27 - 31.20 Mb), a region of homology with human
chromosome 9q34. Here, we will summarize the status of our ongoing candidate
gene analysis for ebo. Positional candidate genes include Rapgef1, Slc27a4,
Coq4, Gle1, Spna2, Zdhhc12, Zer1, Tbc1d13, Ccbl1, and Nup188.
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MOLECULAR GENETIC ANALYSIS OF THE NADPH OXIDASE OF
THE VESTIBULAR SYSTEM
Catrina A Spruce1, John P Flaherty2, Heather E Fairfield1, and David E
Bergstrom1
1
2
New York College of Osteopathic Medicine of New York Institute of
Technology, Old Westbury, NY USA
Age-related vestibular decline is a prevalent and increasingly common ailment
within the American population. Much of the clinical spectrum is due to the loss
and/or displacement of otoconia within the inner ear. Otoconia are proteinaceous
particles containing crystalline inorganic calcite. Affixed to underlying
stereocilia within the vestibular portion of the inner ear, otoconia act as inertial
masses that shift in response to an organism’s linear movements and to the force
of gravity. To better understand the molecular genetics of otoconial deposition
and maintenance, we have previously explored the molecular genetic basis for
three mouse vestibular mutants named “neuroscience mutagenesis facility 333”
(nmf333), “head tilt” (het), and “head slant” (hslt), each of which displays the
classic phenotype of congenital otoconial agenesis. Together these studies
identified Cyba, Nox3, and Noxo1, respectively, as the dysfunction-causative
genes. Cyba, in addition to its role in the inner ear, is also a known component
of the well-characterized and canonical NADPH oxidase of phagocytic cells.
Moreover, Nox3 and Noxo1 are paralogs of the canonical NADPH oxidase
components, Cybb and Ncf1. Thus, the emerging picture is that of a Cyba, Nox3
and Noxo1-containing inner ear NADPH oxidase with structural parallels to the
well-known phagocytic complex. The critical roll of the inner ear oxidase in
otoconiogenesis and proper vestibular function suggests Cyba, Nox3, and Noxo1
as candidate genes potentially involved in human disease- and age-related
otoconial loss. Moreover, the Nox3-based NADPH oxidase, as a source of
potentially harmful reactive oxygen species, is now under investigation as a
possible factor in drug-, age- and noise-related hearing loss. Our presentation
will describe recent gene trap and gene targeting studies aimed at elucidating the
roles of other NADPH oxidase components in the inner ear.
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ABSTRACT 66
MOUSE MODELS OF COSTELLO SYNDROME
Heather E Fairfield1, John P Flaherty2, Catrina A Spruce1, Leah R Donahue1,
and David E Bergstrom1
1
2
New York College of Osteopathic Medicine of New York Institute of
Technology, Old Westbury, NY USA
Costello Syndrome (CS) is a rare, complex, developmental disorder
characterized by a number of features including— failure to thrive,
characteristic facies, delay in intellectual development, hypertrophic
cardiomyopathy, arrhythmia, and predisposition to both benign and malignant
tumors. Past studies identifying gain-of-function mutations in the HRAS gene as
the basis of human CS, and strong conservation with the mouse ortholog Hras1,
have led to the development of a Gly12Val (G12V) mouse model of CS. To
extend animal modeling studies to include various allelic forms of the disease,
we are developing five additional mouse models of CS. Using a recombineeringbased “knock-in” approach, we have completed construction of five vectors,
each containing a loxP-flanked, neomycin resistance (Neor) selection cassette in
intron 1 of Hras1 and site-directed mutations encoding each of the following
five pathogenic alleles — G12A encoded by GCA, G12A encoded by GCC,
G12S encoded by AGC, G12V encoded by GTA and G12V encoded by GTT.
Four of the five strains have been completed creating animal models for the
G12AGCA, G12AGCC, G12VGTA and G12VGTT forms of the disease. Suppression
of each CS-causative allele by the oppositely transcribed Neor transcript can be
relieved by breeding affected mice to inner cell mass- or germline-specific
“deleter” Cre lines such as Meox2-Cre or Zp3-Cre, respectively. Craniofacial
abnormalities like those seen in human patients are clearly evident. Preliminary
phenotypic assessment also suggests possible neoplasia as well as cardiac and
skin abnormalities. Each strain will be deposited into The Jackson Laboratory
Repository. Together, these five models hold the potential for uncovering allelic
and codon preference influences on development and neoplasia in CS,
identifying CS modifying genes, and dissecting tissue-specific facets of CS
using spatially-controlled Cre driver lines.
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ABSTRACT 67
THE APPLICATION OF HIGH-THROUGHPUT SEQUENCING
TECHNOLOGIES FOR MUTATION DISCOVERY IN THE JACKSON
LABORATORY’S MOUSE MUTANT RESOURCE
Laura G Reinholdt, David E Bergstrom, Muriel T Davisson-Fahey, Cathleen M
Lutz, Michael Sasner, Steven A Murray, Jeff Lake, Steven Rockwood, Leah Rae
Donahue and the MMR team
The application of high-throughput sequencing technologies is revolutionizing
the process of mutation detection. By incorporating these technologies, we in
The Jackson Laboratory’s Mouse Mutant Resource (JAX MMR) are extending
our characterization of new spontaneous mutant strains to the point of causative
gene identification. For over fifty years, the mission of the MMR (and its
predecessors) has been to provide mouse models of human genetic illness to the
scientific community. Through our Mouse Phenodeviant Search process,
atypical mice are identified from among the millions of inbred mice produced
yearly at JAX and isolated. This not only ensures genetic stability within the
originating strain; but also, provides a pool of potential spontaneous mutant
mice for further characterization. After colonies are established, heritability is
proven, and the modes of inheritance are determined; subchromosomal locations
for each mutant locus are established by backcross or intercross in conjunction
with SSLP or SNP genotyping. In place of Sanger-based sequencing of
promising candidate genes from select mutant strains, interval-specific array
capture and Illumina GAIIx-based high-throughput sequencing are now being
incorporated to broaden the scope of mutation detection and dramatically
shorten the time to causative gene identification. A general phenotypic
assessment, cryopreservation of embryos or gametes, and archival of mutant
DNA completes the characterization. Findings for each mutant strain are
disseminated through the scientific literature or on the MMR website at
http://mousemutant.jax.org/. Additional projects are underway to incorporate
whole-exome approaches to spontaneous/induced mutation discovery, reducing
the need for genetic mapping. The presentation will enumerate the many
advantages of studying spontaneous mutant mice and summarize current
resource offerings.
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ABSTRACT 68
THE ROLE OF CAML IN THE INNER EAR
Elizabeth C Bryda1, Nathan T Johnson1, Cynthia L Besch-Williford1, Kevin K
Ohlemiller2, and Richard J Bram3
1
Department of Veterinary Pathobiology, Research Animal Diagnostic
Laboratory, College of Veterinary Medicine, University of Missouri, Columbia,
MO, USA
2
Central Institute for the Deaf at Washington University, Department of
Otolaryngology, Washington University School of Medicine, St. Louis, MO, USA
3
Department of Immunology, Mayo Clinic College of Medicine, Rochester, MN,
USA
Calcium modulating cyclophilin ligand (CAML) is a ubiquitously expressed
cytoplasmic protein which appears to play a role in embryonic and thymocyte
development, EGFR and LCK signaling, and chromosomal stability. In
previous studies, we demonstrated that there is a protein –protein interaction
between CAML and the cytoplasmic region of Cadherin23. This led us to
speculate that CAML might be important in the inner ear and play a role in the
development and/or function of hair cells. To enable spatially and temporally
controlled Caml gene expression, a mouse line was generated that allows
knockout of Caml expression specifically in the inner ear upon administration of
tamoxifen. Using this mouse model, tamoxifen was administered immediately
after birth to neonates or daily to pregnant females from day E10.5 to E15 to
assess the effect of loss of CAML during postnatal or embryonic development
respectively. Hearing in treated animals was tested by auditory brainstem
response (ABR) analysis. The presence of hair cells was evaluated via
histological analysis of the inner ears of treated mice. To date, there is evidence
that lack of Caml expression in the inner ear leads to deafness. Elucidating a
role for CAML in the inner ear will aid our understanding of the molecular
pathways important for auditory development and function.
102
ABSTRACT 69
INTERESTING PHENOTYPES FOUND AS PART OF THE INFECTION
CHALLENGE IN THE WELLCOME TRUST SANGER INSTITUTE’S
MOUSE GENETICS PROGRAMME
Simon Clare, Leanne Kane, Lynda Mottram, Jacqui White, Ramiro Ramirez –
Solis, and Gordon Dougan
All the Mouse genome project team at the Sanger Institute
Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus,
Cambridgeshire, UK
As part of the Wellcome Trust Sanger Institute’s Mouse Genetics Programme all
mutant mouse lines generated in this high throughput programme are challenged
with Salmonella Typhimurium an intracellular pathogen which induces a
systemic disease and Citrobacter rodentium a natural mouse pathogen which
forms attaching and effacing lesions on the surface of the gastrointestinal lumen
(details of the challenges can be found on the poster “Phenotyping of knockout
mice using bacterial pathogens as part of the Wellcome Trust Sanger Institute’s
Mouse Genetics Programme”). To date we have identified 18 genes which
contribute to controlling the susceptibility to bacterial infection out over 250
knockout mice lines phenotyped so far. These phenotypes include hits in novel
genes as well as gene of known function and range from severe phenotypes,
were the mice succumb to infection rapidly, too more subtle phenotypes. Here
we would like to show a selection of the data generated as part of this
programme and details of how to freely access all the data and resources from
the programme as a whole.
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ABSTRACT 70
THE EXPRESSION OF SEPTIN 9 IN HUMAN BREAST CANCER
Diana Connolly1, Zhixia Yang1, Elias T. Spiliotis2, Maria Castaldi3, Nichelle
Simmons3, Pascal Verdier-Pinard4, and Cristina Montagna
1
Department of Genetics, Albert Einstein College of Medicine of Yeshiva
University, Bronx, NY, USA
2
Department of Biology, Drexel University, Philadelphia, PA, USA
3
Department of Surgery, Jacobi Medical Center, Bronx, NY, USA
4
INSERM UMR 911 CR02, Aix-Marseille Université, Marseille, France
Septins are a family of GTPases known for their involvement in various cellular
processes and interactions with the cytoskeleton. Septin 9 (SEPT9) in particular,
is one of the fourteen members of this family that is of particular interest
because of its contribution to breast tumorigenesis. Our group identified SEPT9
as a novel oncogene that is amplified and over-expressed in breast cancer.
Alteration of SEPT9 expression has also been implicated in ovarian carcinoma
and results in cell cycle arrest when down-regulated. The study of SEPT9 is
complex due to the potential ability of this gene to encode 18 different isoform
transcripts and at least 5 different polypeptides. Studies show that SEPT9
isoforms are differentially expressed between normal and tumor breast cells, but
their relation to stages of breast cancer development and breast cancer subtypes
is still unclear. Our group has approached this question by analyzing isoform
expression at the DNA, RNA and protein levels. Using mouse models for breast
cancer progression, and human tumor and matching adjacent normal breast
tissue, we have detected gene amplification and expression changes of the
SEPT9 isoforms among the tissue types. For the first time we could show that
pre-malignant cells have a SEPT9 expression profile that distinguishes them
from both normal breast and advanced adenocarcinomas. Moreover, we found
that the differential expression of at least one isoform (SEPT9_v3) was due to
epigenetic regulation via the methylation status of an alternative promoter
region. Our data suggest that other alternative promoters within this gene could
also be regulated by DNA methylation and thus dramatically affect the
expression of SEPT9 isoforms in tumorigenesis. These findings are supported by
the establishment of a diagnostic SEPT9 DNA methylation assay for detection
of colorectal cancer in peripheral blood, and furthermore indicate that the
amplification and differential expression of SEPT9 isoforms may represent a
potential molecular signature with functional and diagnostic significance for
breast tumor progression and early detection.
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ABSTRACT 71
GENETIC CONTRIBUTION TO LIVER FIBROSIS
David DeSantis1, Michelle Pritchard3, Laura Nagy3, Joseph Nadeau2, and
Colleen M Croniger1
1
Departments of Nutrition, 2Department of Genetics, School of Medicine, Case
Western Reserve University, Cleveland, Ohio, USA
3
Department of Gastroenterology, Department of Pathobiology, Cleveland
Clinic, Cleveland, Ohio, USA
Liver disease is one of the serious complications associated with either chronic
excessive alcohol consumption or calorie consumption. Complications can range
from steatosis to hepatitis and eventually fibrosis and cirrhosis. Pathological
evolution of alcoholic steatohepatitis (ASH) and non-alcoholic steatohepatitis
(NASH) are very similar yet only 20-50% of these patients develop cirrhosis.
The mechanism for progression to fibrosis in these patients is presently
unknown. Current nutritional and transgenic models of hepatic steatosis
replicate preconditions for ASH and NASH, but fail to represent the
multifactorial pathogenesis of these diseases. To identify susceptibility genes to
fibrosis, we have analyzed the response to high fat high sucrose diet (HFHS) in
chromosome substitution strains (CSS) developed by Dr. Joseph Nadeau (Case
Western Reserve University). CSS strains have one chromosome from the A/J
inbred strain that has been substituted for the corresponding chromosome on the
B6 genetic background. CSS-17 which contains A/J Chromosome 17 was
resistant to obesity and steatosis. Analysis of congenic strains derived from
CSS-17 identified a very small segment of A/J chromosome that conferred
resistance to obesity and steatosis. In collaboration with Dr. David Adams (The
Wellcome Trust Sanger Institute), A/J Chromosome 17 has been deep short-read
sequenced to 22X coverage. Using comparative analysis of the sequence from
A/J and B6 Chromosome 17, we have identified genes in the small region of A/J
chromosmone that have single nucleotide polymorphisms (SNPs) in the
proximal region or coding region. We hypothesize that these genes play a
critical role in development of hepatic fibrosis. Our preliminary data support this
hypothesis. To induce fibrosis we have used CCl4 administration in A/J mice,
CSS17 and congenic strains from CSS17. A/J mice remained resistant to CCl4induced injury while CSS-17 and B6 mice developed CCl4- induced injury and
fibrosis. Most importantly the congenic strain containing only 27.8 Mb of A/J
chromosome had CCl4- induced fibrosis at significantly higher levels compared
to B6 mice. A region more distal was protected from CCl4-induced fibrosis.
Thus Chromosome 17 contains genes that are contributing to susceptibility to
CCl4 liver injury.
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ABSTRACT 72
DIET, OBESITY, AND SUSCEPTIBILITY TO COLON POLYPS
Stephanie K. Doerner 1,3 and Joseph H. Nadeau 1,2,3
1Department of Genetics, Case Western Reserve University School of Medicine,
Cleveland, Ohio, USA
2Case Comprehensive Cancer Center, Case Western Reserve University,
Cleveland, Ohio, USA
3Case Center for Transdisciplinary Research on Energetics and Cancer
Colon cancer is the third most common cancer worldwide and is the second
leading cause of cancer related deaths in the United States. Both genetic and
environmental factors such as diet, obesity, microbial profile, inflammatory
diseases and heritability contribute to susceptibility. However, assessing the
contributions of obesity and diet on the development and progression of colon
cancer is difficult because they are strongly correlated in humans. Recent work
from our laboratory suggests that the amount of dietary saturated fat plays a
critical role in the development of intestinal polyps in the B6.ApcMin/+ mouse
model. Using Congenic-Consomic strains, we show that a high-fat diet,
regardless of susceptibility to diet-induced obesity, affects polyp number. We
show that the type of fat (saturated versus omega-6 polyunsaturated)
significantly affects polyp number after 60 days. We also show that polyp
numbers stabilize in mice after 30 days on the high-fat and low-fat diets.
Preliminary data suggests that pro-inflammatory cytokines are elevated in
intestinal polyps of mice fed the high-fat diet compared to the low-fat control
diet and that circulating inflammatory factors are also elevated in mice fed a
high-fat diet. Together, our diet studies demonstrate that the quantity and kind of
dietary fat influences polyp number, independent of obesity. Although much has
been learned about the effects of diet on cancer severity, the mechanism remains
unknown. The B6.ApcMin/+ mouse model for colon cancer will be used to test the
effects of alternative fat compositions on cancer severity, the influence of
inflammation in carcinogenesis, and the interaction between diet and WNT
signaling.
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ABSTRACT 73
GENETIC REGULATION OF FRACTURE RISK IN INBRED MICE
Leah Rae Donahue, GA Churchill, S-W Tsaih, and WG Beamer
The Jackson Laboratory, Bar Harbor, Maine, USA
The measurement often used to predict bone strength is bone mineral density
measured by x-ray absorptiometry. Other bone traits - size, shape, architecture
and material properties, as well as muscle mass - are important qualitative
contributors to bone strength and should be considered when predicting fracture
risk. There is intense interest in genetic determinants of fracture risk based on
these indices of bone strength. Interpretation of human studies is complicated by
heterogeneity and environmental differences, indicating that genetic regulation
of bone strength is complex and that animal models are critical to progress in
this field. Our goal was to determine which muscle and bone phenotypes predict
femoral bone strength in mice and to discover genetic models for further
analyses of factors that contribute to fracture risk. We collected skeletal
geometry, muscle mass, volumetric BMD (vBMD), and bone strength data from
female mice of 9 inbred strains. pQCT (SA Plus,Stratec) was used to measure
vBMD, periosteal perimeter (Ps.pm), and muscle area at the mid diaphysis;
digital calipers were used to measure femur length and medio-lateral diameter
(M/L) at mid diaphysis; peak load, stiffness, and energy to break (EB) were
measured by three point bending (MTS). EB is often used as a measure of the
amount of energy required to cause bone to fracture. We found that peak load
and stiffness were both predicted by femur length (p=.002;.001), M/L diameter
(p=.009;.029), muscle area (p=.001;.011), and vBMD (p=006;.002). EB was
predicted by Ps.pm (p=.044), M/L (p=.037), and muscle area (p=.009), but was
independent of vBMD. A strategy to genetically decompose the EB phenotype
could be based on crosses between a low EB (low bone size/muscle area) strain
and a high EB (plus high bone size/muscle area) strain. Such a combination is
present in SWR/J and FVB/NJ, or in 129/SvImJ crossed to C57BL/6J. The F2
generation progeny from either cross would provide mice that could be used to
analyze the genetic regulation of EB. These data show that bone geometry,
density, and muscle mass are important determinants of femoral strength, and
illustrate that bone strength is a complex trait with genetic regulation. We
propose a mouse genetic model to discover the genetic regulation bone
geometry, muscle mass, and BMD, and to determine the relative contribution of
each to overall bone strength and fracture risk.
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ABSTRACT 74
IMPRINTED XLAS: A NEW PLAYER IN BONE AND ADIPOCYTE
METABOLISM
Sally Eaton, Simon Ball, Colin Beechey, Christine Williamson, and Jo Peters
MRC Harwell, Mammalian Genetics Unit, Harwell Science & Innovation
Campus, Oxfordshire, OX110RD, United Kingdom
It is recently understood that bone and energy metabolism are tightly coregulated through the central and sympathetic nervous systems. Now that this
mechanism of the control of bone metabolism has been described we are better
placed to understand how new entrants regulate both bone and adipocyte
metabolism. We describe here a new mouse mutation which presents with a
severe growth retardation, a reduction in fat mass, an increased metabolic rate, a
lowered bone mineral density, an increase in Ucp1 and a reduction in leptin. We
further describe that this phenotype can be attributed to truncation of the
paternally expressed transcript, Gnasxl resulting in loss of the protein, XLAS.
Gnasxl arises from the imprinted Gnas cluster and is expressed exclusively from
the paternally derived allele. Gnasxl encodes for a protein that acts as the alpha
subunit of the heterotrimeric Gs protein and regulates receptor-stimulated cAMP
production. This is to our knowledge the first report describing a role of XLAS
in bone metabolism and we propose that XLAS is involved in the co-regulation
of bone and adipocyte metabolism at the level of the hypothalamus through its
actions on the sympathetic nervous system.
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ABSTRACT 75
THE
SANGER
MOUSE
GENETICS
PROGRAMME: HIGH
THROUGHPUT RECESSIVE LETHALITY SCREEN
Jeanne Estabel, Elizabeth Tuck, Damian Carragher, Jennifer Salisbury, and
Jacqueline K. White on behalf of the Mouse Genetics Programme
The Sanger M.G.P., Wellcome Trust Sanger Institute, Genone Campus, Hinxton,
Cambridgeshire, United Kingdom
The Sanger Institute Mouse Genetics Programme aims to make a significant
contribution to the understanding of the function of genes and their role in
disease by generating, characterising and archiving 200 lines of knockout mice
per year. In addition to a standardised battery of primary phenotypic testing
relevant to key disease areas including diabetes, obesity, hearing and vision
disorders, immune disorders, pain and motor function, one key aspect of
understanding the function of genes is to identify their expression profile. The
presence of the β-Galactosidase reporter gene, expressed under the control of the
endogenous promoter of the target gene, makes wholemount expression
profiling in the adult and at various developmental stages possible.
Furthermore, ~31% of the knockout lines of mice assessed to date display a
recessive lethality phenotype. To explore the underlying developmental defects,
we are collecting and assessing embryos from heterozygous intercrosses at
E14.5. Here we report the strategy that we have implemented to facilitate the
efficient, high throughput, large scale expression profile and recessive lethality
analysis of knockout lines, including a tiered approach that we have to document
and interrogate the data. This systematic process involves husbandry
management, embryo malformation assessment and record keeping. We present
a summary of recessive lethality data available to date, and examples of novel
findings for a subset of interesting mutant lines.
109
ABSTRACT 76
MULTIPLE FOREBRAIN CIS-REGULATORY ELEMENTS AT THE
DLX1/2 LOCUS: REDUNDANT FUNCTION OR COMPLEX CONTROL
MECHANISMS?
Marc Ekker, Luc Poitras, Man Yu, Noel Ghanem, and Lisa Tran
Center for Advanced Research in Environmental Genomics, Department of
Biology, University of Ottawa, Canada
The Dlx homeobox genes play an important role in the development of the
vertebrate forebrain. DLX function is essential for the migration and
differentiation of GABAergic interneuron precursors. Of the three Dlx bigene
clusters, the Dlx1/Dlx2 and Dlx5/Dlx6 genes are expressed in the forebrain with
patterns suggesting concerted control mechanisms. We have identified four cisacting regulatory elements (CRE) with forebrain enhancer activity. Of the four
CREs, URE2, and I12b are found at the Dlx1/Dlx2 locus whereas I56i and I56ii
are e located in the Dlx5/Dlx6 intergenic region. The CREs are highly conserved
amongst distant vertebrates, structurally and functionally. However, the four
enhancers diverge markedly in sequence although they target gene expression
with partially overlapping patterns. Immunohistochemical analyses revealed that
the mouse URE2 and I12b/I56i are active in distinct populations of progenitor
cells that will be give rise to different subtypes of adult GABAergic
interneurons. We produced mice with targeted deletion of the I12b CRE. Mice
homozygous for the mutation show partial reductions in Dlx1 and Dlx2
expression in the E13.5 forebrain as well as small increases in the levels of
Mash1 transcripts. They did not display overt developmental abnormalities and
newborn pups were viable. These results suggest partial functional redundancy
of this Dlx CRE at least in subset of GABAergic interneurons and their
precursors. Supported by CIHR MOP14460
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ABSTRACT 77
NOVEL MUTATION DISCOVERY ON ENU MOUSE MUTAGENESIS
BY NEXT-GENERATION SEQUENCER
Ryutaro Fukumura, Hayato Kotaki, Yuichi Ishitsuka, Takuya Murata, Shigeru
Makino, Yuji Nakai, and Yoichi Gondo
Mutagenesis and Genomics Team, RIKEN BioResource Center, Japan
We developed a reverse genetics tool to provide allelic series of point mutations
in any mouse genes. We have produced about 10,000 Generation-1 (G1) male
mice derived from ENU-treated C57BL/6J males. We have preserved them as
frozen sperm and extracted each genomic DNA for the mutation screening of
target genes. Thus, the dual archives of frozen sperm and genomic DNA are
considered to be the Mutant Mouse Library. We have called this system the
RIKEN Gene-Driven Mutagenesis System (RGDMS). By using various
methods to detect novel point mutations, we have estimated the ENU-induced
mutation rate is ~1/1,000,000bp in a G1 genomic DNA sample. It implies that
one G1 mouse has ~3,000 mutations. Therefore, a total of 30,000,000 mutations
are reserved in the RIKEN Mutant Mouse Library. We have made the RIKEN
Library open to research community and have been finding ~100 ENU-induced
mutations per year in various target genes based upon the requests. To
significantly enhance the mutation detection efficiency, we have started to resequence the G1 mouse genomic DNAs in the library by the next-generation
sequencers. The shotgun sequencing of the whole mouse genome by the
available next-generation sequencers has not been cost effective yet, thus we
target protein-coding sequences to find the mutation. First, we selected 10
chromosomal regions and extracted about 4Mb exon-sequences. We analyzed
them by illmumina GAIIx (two G1 genomes; 1675 and 381) and by AB
SOLiD3plus (three G1 genomes; 1675, 381 and 15373). We estimated the
mutation detection efficiency with known 57 SNPs on Chr 8 between DBA/2J
and C57BL/6J as positive controls. The mutation detection efficiency was more
than 90% by both systems. We have so far found 20 and 7 novel ENU-induced
mutations from targeted exon-sequences and non-targeted regions on three G1
genomes, respectively. Eight and one mutations were missense and nonsense
mutations, respectively.
111
ABSTRACT 78
THE
SANGER
MOUSE
GENETICS
PROGRAMME: HIGH
THROUGHPUT CHARACTERISATION OF KNOCKOUT MICE FOR
GENES INVOLVED IN GLUCOSE METABOLISM
Anna-Karin Gerdin, L Roberson, Jacqueline K White on behalf of the Mouse
Genetics Programme
The Sanger Mouse Genetics Programme, Wellcome Trust Sanger Institute,
Hinxton, Cambridgeshire, United Kingdom
The Sanger Mouse Genetics Programme aims to make a significant impact on
our understanding of the function of genes and their role in disease, by
generating, characterising and archiving 200 knockout lines per year. To date
knockout mice for more than 200 genes have been characterised using a battery
of primary phenotyping assays relevant to key disease areas including diabetes,
obesity, hearing and vision disorders, immune disorders, pain and motor
function. Expression profiling via the lacZ reporter gene is performed for each
mutant line in adults and at E14.5. We will describe the phenotypic
characterisation of seven knockout lines for genes involved in glucose
metabolism. The genes we will focus on are Gys2, Gsk3a, Akt2, Pfkl, Tpi1, Ldha
and Ide. We will explore how the data generated by the primary screen might be
used to further the understanding of the interplay of genes and disease, and how
it provides insight into the underlying biological pathways. Our data provides
support for some previously published studies but also adds significant novel
information to the scientific community. The tests used to investigate the
metabolism of these mice include high fat diet exposure, body weight curve,
indirect calorimetry, glucose tolerance test, body composition examination, core
body temperature and clinical chemistry as well as expression profiling and
tissue collection to our Biobank.
112
ABSTRACT 79
EUROPHENOME:
LARGE
DATASET
VISUALISATION,
STATISTICAL ANALYSIS AND DATA EXPLORATORY TOOL FOR
MOUSE PHENOTYPING DATA
Ahmad Hassan, Hugh Morgan, Andrew Blake, Simon Greenaway, The
EUMODIC Consortium, John M. Hancock, and Ann-Marie Mallon
Medical Research Council, Mammalian Genetics Unit, Harwell Science and
Innovation Campus, Oxfordshire, OX11 0RD, UK
A major aspect of medical research is to utilise results to understand disease and
improve human health. In recent years, a lot of effort has been made in medical
science in linking the genotype data with phenotype data. The correlation of
genotype with phenotype information enables a better understanding of human
diseases and phenotype alterations that results from genetic changes. The
EuroPhenome project (http://www.EuroPhenome.org) is a mouse phenotyping
data repository that hosts the raw and annotated high-throughput phenotyping
data arising from different projects such as European Mouse Disease Clinic
(http://eumodic.org). EUMODIC is gathering data from the EMPReSSslim
pipeline (http://www.empress.har.mrc.ac.uk) which is performed on inbred
mouse strains and knock-out lines arising from the EUCOMM project.
Europhenome provides the tools to visualise mouse phenotype and genotype
data in an integrated manner. In addition, it enables the phenotyping data to be
viewed in variety of ways i.e. Graphical representation of phenotyping data for
inbred and mutant strain, graphical representation of statistically significant
phenovariant data, mouse phenotype (MP) ontology tree and access to the data
via web services. The raw phenotyping data captured in EuroPhenome is
annotated by an annotation pipeline which automatically identifies statistically
different mutants from the appropriate baseline and assigns ontology terms for
that specific test. The mutant phenotypes can be viewed using two
EuroPhenome tools: PhenoMap, a graphical representation of statistically
relevant phenotypes, and mining for a mutant using ontology terms. To assist
with data definition and cross-database comparisons, phenotype data is
annotated using combinations of terms from biological ontologies.
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ABSTRACT 80
THE INTRAGENE RESSOURCE CENTRE, A NATIONAL CENTRE
FOR MOUSE GENETICS, DISTRIBUTION, ARCHIVING AND
FUNCTIONAL IMAGING
Cecile Fremond, Stephanie Lerondel, Christelle Martin, and Yann Herault
Transgenesis, Archiving of Animal Models, CNRS, TAAM, UPS44, Institut de
Transgenose, 3B rue de la Ferollerie, 45071 Orleans cedex2, France
The INTRAGENE Ressource Centre provides the scientific community with all
the technical facilities essential for creating, maintaining, distributing,
conserving and analyzing transgenic murine models, either for the study of
human diseases, or for fundamental modelling of genetic analysis in physiology,
development, immunology, neurobiology, etc... Its main activities are associated
to the Transgenesis, Archiving of Animal Models service unit from the CNRS
based in Orleans and Villejuif. The Resource Center creates new transgenic
models, mainly on the Villejuif site in the SErvice for Transgenic Animals
(SEAT) while on its Orleans site, it hosts the Centre for Distribution,
Transgenesis and Archiving (CDTA), a "mouse repository", which represents a
large collection of mutant and transgenic mouse strains available on request
from, or in agreement with, the EMMA network (www.emmanet.org).
Furthermore the CDTA offers a large set of zootechnical services to the
scientific community including rederivation, sanitary and genetic controls and
gnotoxenisation. Finally, the CIPA department is devoted to small animal
imaging with a large set of complementary imaging techniques. The operational
phenotyping analysis and different services fulfill the recommendations set out
by the EUMORPHIA program and also to ensure appropriate animal ethics and
welfare. The Resource Center has been recognized since 2001, by way of an
accreditation French Infrastructure in Biology, Health and Agronomy, and since
then has been an integral part of the TGIR department of the Life Science
Ministry: CELPHEDIA, and as a partner of the Infrafrontier European funded
consortium. It is moving to the large scale infrastructure for Life Science and
Biotechnology to answer further to the needs of the scientific community in
mouse models for fundamental research and human diseases.
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ABSTRACT 81
THE DOWN SYNDROME AS A COMPLEX TRAIT RESULTING FROM
THE INTERACTION OF DOSAGE SENSITIVE GENES UNRAVELLED
IN THE MOUSE MODEL
V Brault1, A Duchon1, E Dalloneau1, P Lopes1, I Abizanda2, S Luiller3, JC
Bizot3, C Borel4, M Raveau1, D Marechal1, J Lignon1, S Pothion2, F Trovero3, V
Tybulewicz5, EM Fisher5, S Antonarakis4, M Dierssen2, and Yann Herault1,7,8
1
IGBMC, CNRS, INSERM, UdS, UMR7104, U964, Illkirch, France
2
Genes and Disease Program, Center for Genomic Regulation, Dr Aiguader 88,
08003 Barcelona, Spain and CIBER de Enfermedades Raras CIBERER,
Barcelona, Spain
3
Key-Obs S.A., Allée du Titane, Orléans, France
4
Department of Genetic Medicine and Development, University of Geneva
Medical School, 1 Rue Michel-Servet, 1211 Geneva, Switzerland
5
MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London
UK
6
Department of Neurodegenerative Disease Institute of Neurology, London, UK
7
CNRS UPS44 TAAM, Orleans, France
8
ICS, ILLKIRCH, France
More than 50 Years after the discovery of the Trisomy 21, the underlying
genetic basic of the Down syndrome (DS), is still a challenge. Human genetic
studies clearly demonstrated the complex genotypes - phenotypes relationship in
DS with several loci participating to the DS traits. The current hypothesis is
based on interactions between “dosage sensitive” genes or regions spread along
the Hsa21 that are responsible for the complex feature of the pathology and
could explain the penetrance and the variability of the traits. Currently a little of
this loci has been identified. In order to go further and to decipher the gene
interactions generating the DS phenotype, we created new partial trisomies and
monosomies in the mouse for the different regions homologous to Hsa21 by
using chromosomal engineering. Orthologuous genes on Hsa21 are located on
mouse Chromosomes (Mmu) 16, 17 and 10. The Ts65Dn well-known mouse
models for DS that is trisomic for part of the Mmu16 homologous region and
exhibits many of the characteristics of DS has been a key model for the better
understanding of the disease and the development of pharmacological
intervention (Fernandez et al. Nat Neurosci 2007, 10, 411-413). Interestingly
several new reports for larger trisomies, including the Hsa21 transchromosomic
mice, Tc1 (O'Doherty et al., Science 2005, 309, 2033-2037) and the complete
trisomic model developed by Yu and colleagues (Yu, T. et al.., Hum Mol Genet
2010, 19, 2780-2791) demonstrate that increasing the gene number in trisomic
model could also result in the masking of specific phenotypes. Using our series
of mouse trisomic and monosomic models, we deciphered the contribution of
115
several regions in the induction of DS phenotypes and identified several dosage
sensitive genes controlling behavior, learning and memory, cardiovascular,
craniofacial morphology and respiratory function. Now we are looking at the
pathways that are affected, with the hope that this would lead to development of
new therapeutic approaches. We will report here the phenotypic analysis of the
new models and the results obtained on several traits including cognition,
learning and memory and additional phenotypes. Our data support the current
hypothesis that the DS phenotype is a complex trait with several dosage
sensitive loci found on Hsa21, impacting the central nervous system and other
organs, that interact together. A few of those loci and the pathways that are
modified in DS will be discussed.
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ABSTRACT 82
THE EUROPEAN MOUSE MUTANT ARCHIVE (EMMA)
Michael Hagn6, G Tocchini-Valentini1, Y Hérault2, S Brown3, U Lendahl4, J
Demengeot5, M Hrabé de Angelis (EMMA Director)6, E Birney7, R RamirezSolis8, JL Mandel9, and L Montoliu10
1
Consiglio Nazionale delle Ricerche, Istituto di Biologia Cellulare (CNR-IBC),
Monterotondo Scalo, Italy
2
Centre National de la Recherche Scientifique, Transgénèse et Archivage
d’Animaux Modèles (CNRS-TAAM), Orleans, France
3
Medical Research Council, Mammalian Genetics Unit (MRC-MGU), Harwell,
UK
4
Karolinska Institutet, Department of Cell and Molecular Biology (KI-CMB),
Stockholm, Sweden
5
Fundação Calouste Gulbenkian, Instituto Gulbenkian de Ciência, Oeiras,
Portugal
6
Helmholtz Zentrum München, Institute of Experimental Genetics (HMGUIEG), Neuherberg, Germany
7
European Molecular Biology Laboratory, European Bioinformatics Institute
(EMBL-EBI), Hinxton, UK
8
Genome Research Limited, Wellcome Trust Sanger Institute (WTSI), Hinxton,
UK
9
GIE-Centre Européen de Recherche en Biologie et en Médecine, Institut
Clinique de la Souris (GIE-CERBM-ICS), Illkirch, France
10
Consejo Superior de Investigaciones Scientificas, Centro Nacional de
Biotecnología (CNB-CSIC), Madrid, Spain
The European Mouse Mutant Archive (EMMA) offers the worldwide scientific
community a free archiving service for its mutant mouse lines and access to a
wide range of disease models and other research tools. A full description of
these services can be viewed on the EMMA website at
http://www.emmanet.org. The EMMA network is comprised of ten partners who
operate as the primary mouse repository in Europe and is funded by the
participating institutes and the European Commission FP7 Capacities Specific
Program. EMMA’s primary objectives are to establish and manage a unified
repository for maintaining mouse mutations and to make them available to the
scientific community. In addition to these core services, the consortium can
generate germ-free (axenic) mice for its customers and also hosts courses in
cryopreservation. All applications for archiving and requests for mutant mouse
strains are submitted through the EMMA website. Mouse strains submitted for
archiving are evaluated by EMMA’s external scientific committee. Once
approval has been granted depositors are asked to send mice of breeding age to
one of the EMMA partners for embryo or spermatozoa cryopreservation. Strains
117
held under the EMMA umbrella can be provided as frozen materials or rederived and shipped as live mice depending on the customer’s needs. However,
certain strains that are in high demand are maintained as breeding colonies to
facilitate their rapid delivery. All animals supplied by EMMA are classified as
SPF in accordance with the FELASA recommendations. EMMA is a founding
member of FIMRe (International Federation of Mouse Resources) and actively
cooperates with other leading repositories like TJL and the MMRRC in the US
and BRC RIKEN from Japan.
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ABSTRACT 83
BROAD BASED PHENOTYPING APPROACHES FOR THE
CHARACTERISATION OF MUTANT MOUSE LINES IN EUMODIC
AND THE HARWELL AGING SCREEN
Heena Vanmalibhai Lad, KA Lee, A Blake, H Morgan, A Hassan, S Greenaway,
AM Mallon, HJ Gates, SDM Brown, and The EUMODIC Consortium
MRC Harwell, Harwell Science and Innovation Site, Oxfordshire, UK
Central to successfully characterising the function of every gene in the mouse
genome is the use of broad based phenotyping screens that will capture a range
of phenotypes across mutant mouse lines. In the EUMODIC consortium, in
order to capture robust and reliable phenotype information we have adopted and
developed validated Standard Operating Procedures (SOPs), each which has the
potential to reveal a variety of phenotypic perturbations underlying the genetic
variant of interest. The SOPs are combined into the EMPReSSslim phenotyping
platform. EMPReSSslim comprises two main strands of phenotyping that form
‘Pipeline 1’ and ‘Pipeline 2’, each designed to minimise the impact that
preceding and successive protocols may have on the phenotypic outcome. Data
combined from these pipelines reveals aspects of: dysmorphology; metabolism
and physiology; bone structure and density; behaviour; sensory systems; and
immunology. Complementary to the rigorous screening of mutant lines is the
cataloguing of the phenotypes in a standardised format that will allow these data
to be linked to ontological terms. In EUMODIC, standardisation of data capture
has been achieved through devising a set of defined parameter lists for each
SOP. Phenotypic ontologies are used to identify phenodeviants where a
parameter for the mutant is statistically different to the control value. These are
displayed in a heatmap (www.europhenome.org/ontologybrowser/heatmap.php),
called ‘PhenoMap’, for each of the mutant lines. Adjusting the significance
level and the effect size determines the colour displayed and thereby its related
ontologies. For the data display and mark-up process to be operational, each of
the components contributing at the successive levels is precisely mined to index
the information for each mutant by pipeline or phenotype(s) of interest. Using a
similar approach and many of the tests included within the EUMODIC
pipelines, a large aging screen is underway at Harwell on mutant lines generated
from the ENU programme. The main objective of the undertaking will be to
identify the severity of the phenotype(s) and its associated genetic pathways
with respect to late onset and/or age-related diseases.
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ABSTRACT 84
FBXO11 REGULATES TGF BETA SIGNALING THROUGH TRP53
Hilda Tateossian, Susan Morse, and Steve DM Brown
MRC Mammalian Genetics Unit, Harwell, Oxfordshire, UK
The Jeff mouse mutant develops deafness due to chronic proliferative otitis
media. The gene mutated in Jeff was identified as Fbxo11, a member of the Fbox family (Hardisty et al. 2006, Hum. Mol. Genet. 15: 3273). We previously
reported that Fbxo11 affects TGF beta signaling by regulating the levels of
phospho-SMAD2 in the epithelial cells of palatal shelves, eyelids and airways of
the lungs (Tateossian et al. 2009, PathoGenetics, 2:5, 1-14). FBXO11 has
recently been identified as a NEDD8 ligase for the tumor suppressor protein p53
in human colorectal carcinoma cells. It has been also reported that FBXO11 can
inhibit TP53 activity without affecting its stability (Abida et al. 2007, J.
Biochem Chem., 282, 1797-1804). Given the cross-talk between TGF beta
signalling and p53, and the reported interaction between FBXO11 and TP53, we
proceeded to examine the genetic and biochemical interactions of Fbxo11 and
Trp53 in the developing mouse. To study the genetic interaction we crossed
mice heterozygous for Fbxo11 to homozygous Trp53 knock-outs. The adult
double heterozygote mice are smaller than Trp53 heterozygotes and their palates
are not properly fused. The double homozygote new born mice also appear
underdeveloped compared to their littermates. The phenotype of the double
mutants indicates a genetic interaction of Fbxo11 and Trp53. To confirm the
physical interaction between FBXO11 and TRP53 in developing tissues we used
an immunoprecipitation strategy. Total protein from E15.5 lungs was
immunoprecipitated with FBXO11 antibody and the Western blot analysis
revealed that the antibody precipitated TRP53 as well as FBXO11. Furthermore
TRP53 appears NEDD8-conjugated when complexed with FBXO11.
Immunoprecipitation with TRP53 also confirmed that the two proteins are in a
complex. Moreover, we have shown that only one isoform of FBXO11 interacts
with TRP53. It is known that TRP53 is a partner of SMAD2 in the activation of
multiple TGF beta target genes in mammalian cells and Xenopus embryos
(Cordenonsi et al. 2003, Cell, 113, 301-314). We are currently investigating if
the FBXO11-SMAD2-TRP53 complex in the developing mouse affects
expression of genes under the joint control of TRP53 and TGF beta. Further
studies into the interacting partners of FBXO11 and the signaling pathways it
impacts will improve our understanding of the molecular mechanisms of middle
ear inflammatory disease.
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ABSTRACT 85
THE DEVELOPMENT OF THE INTEGRATED DATABASE OF
MAMMALS IN RIKEN
Hiroshi Masuya1, Yuko Makita2, Koro Nishikata2, Yuko Yoshida2, Terue
Takatsuki1, Kasunori Waki1, Nobuhiko Tanaka1, Yoshiki Mochizuki2, Norio
Kobayashi2, Riichiro Mizoguchi, Teiichi Furuichi4, Hideya Kawaji5, Daub
Carsten5, Yoshihide Hayashizaki5, Shigeharu Wakana1, Atsushi Yoshiki1, Kaoru
Fukami-Kobayashi1, and Tetsuro Toyoda
1
RIKEN BioResource Center, Tsukuba, Japan
2
RIKEN BASE, Yokohama, Japan
3
Department of Knowledge Systems, ISIR, Osaka University, Ibaraki, Japan
4
Laboratory of Molecular Neurogenesis, RIKEN BSI, Wako, Japan
5
RIKEN OSC, Yokohama, Japan
To secure the sustainability, accessibility, utility and publicity of the data
produced from multiple large-scale programs, RIKEN has developed an official
integrated database for mammal data. The database integrates not only RIKEN’s
original databases, such as FANTOM, ENU mutagenesis program and RIKEN
Cerebellar Development Transcriptome Database (CDT-DB) and BRC Mouse
Web Catalog, but also imported public databases such as Ensembl, MGI and
OBO ontologies. Our integrated database has been constructed on RIKEN’s
semantic-web-based cloud system termed SciNeS or Scientists’ Networking
System, where the data and metadata are structured as a semantic web and are
downloadable in various standardized formats. The outstanding feature of this
database is the integrated implementation of the general knowledge (i.e. “allele
is a variant of a genetic marker”) represented in the top-level ontology, Yet
Another More Advanced Top-level Ontology (YAMATO) and public
ontologies, and the instantiated knowledge in databases (i.e. “a datum for an
ENU induced allele has a informational relationship to a gene recode in the MGI
database”). This will provides 1) the dissemination of RIKEN’s data to be
coordinated (or annotated) with public meta-information to increase its
accessibility and 2) comprehensive, sustainable and cost-effective data
management and maintenance across multiple databases. The Sustainability of
database becomes a major issue on the bioinformatics field. Therefore, by this
database, we would like to propose one of the generally useful and cost-effective
ways to solve this problem.
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ABSTRACT 86
COMBINING CLINICAL AND EXPRESSION QTLS IN THE CONTEXT
OF INFECTION: NEW INSIGHTS INTO SUSCEPTIBILITY TO
INFLUENZA
Gregory A. Boivin1,2, Julien Pothlichet2, Emil Skamene2, Earl G. Brown 3,
Robert Sladek1,4,5, and Silvia M. Vidal 1,2,6
1
Department of Human Genetics, McGill University, Canada
2
McGill Centre for the Study of Host Resistance, McGill University, Canada
3
Department of Biochemistry, Microbiology and Immunology, University of
Ottawa, Canada
4
Genome Quebec Innovation Center, Montreal, Canada
5
Department of Medicine, Experimental Medicine Division, McGill University,
Canada
6
Department of Microbiology and Immunology, McGill University, Canada
Seasonal influenza infections cause millions of severe cases and over 250,000
deaths annually worldwide. Influenza virus also causes recurrent pandemics in
humans (e.g., 1918 [over 20 million deaths], 1957 [~2 million deaths], 1968 [~1
million deaths]). The large collection of influenza viruses circulating in animal
species (including 16 “H” subtypes and 9 “N” subtypes as classified by surface
cell receptors) makes the prediction of causal strains of future pandemics and
epidemics difficult. Further, the high mutation rate inherent in influenza virus
replication makes the timeline of future influenza outbreaks hard to predict. By
studying immunological responses against influenza in the host species, it may
be possible to discover common mechanisms of susceptibility in response to
many influenza strains. This will ideally lead to quickly implementable therapies
with wide clinical relevance, reducing economic and health burdens of future
influenza epidemics and pandemics. We have created a mouse model of highly
pathogenic influenza (hp-flu), which reproduces the hallmark inflammatory
response and over-expression of cytokines associated with susceptibility to hpflu in humans by using a mouse adapted strain of influenza (A/HK/1/68-MA20
[H3N2]). By studying a panel of 34 closely related mouse strains created from
susceptible A/J and resistant C57BL/6 mice (the AcB/BcA set of recombinant
strains) we identified four loci strongly associated with altered influenza
susceptibility, which range in size from 6 to 30 Mb. To more efficiently identify
causative genes underlying these loci, we incorporated expression profiles
obtained from uninfected lung tissue of 54 AcB/BcA mice. Functional
characterization was performed for a subset of genes showing evidence of cisregulation. These co-localized clinical and expression QTLs have confirmed a
previously reported influenza susceptibility locus and have identified a novel
mechanism of susceptibility to influenza virus infection related to lipid
biology. This study represents a potential road map for future studies attempting
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to incorporate expression QTLs with traditional forward genetics towards
efficient gene discovery in the context of infection.
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ABSTRACT 87
A NATURE OF THE BLASTOMER DNA METHYLATION
DETERMINATION
Andrey A. Ivanov
Vernadsky Institute of Geochemistry and Analytical chemistry RAS, Moscow,
Russia
All known biochemical processes involves the fractionation of isotopes [1]. In a
separate series of the molecular biology experiments devoted to studies and
simulation modeling on the metabolism environment isotopy, a phenomenon of
a non-equal methylation of the isotopically different DNA copies has been
found. It seems that the nature of this phenomenon somehow relates to a change
in the DNA double helix conformational status [2]. This phenomenon is no
doubt significant for making perfectly clear the very nature of the blastomers
functional determination. This indicates to the zygote-specific, universal for
zygote as well as for a whole biodiversity range, fundamental regularity. A
regularity that consists in a non-random, truly regular, distribution of the
isotopically different nucleotide forms among the blastomer DNA
polynucleotide pairs. Noteworthy, the free nucleotides pool in any zygote
originally contain approx. 10% of the isotopically heavy (Carbon-13 enriched)
nucleotides. But in a course of closed, self-isolated, zygote cleavage process –
there is no regeneration (no “refilling”) of the free nucleotides pool occurred.
The isotopically light nucleotides are the most mobile ones, so they are about to
get involved into a DNA synthesis more actively compared to the isotopically
heavy ones. That’s why the zygote cleavage kinetic - from one stage to the next
one - steadily changes the ratio of isotopically different nucleotide forms to a
favor of the heavy ones. Owing to this, the DNA isotopy patterns should be
different at different zygote cleavage stages. Particularly, this difference comes
up to its maximal level by the zygote cleavage terminal stages. Then, due to
depletion of the zygote free nucleotides pool, a C-13 isotopic self-organization
takes place within the DNA double helix forming polynucleotide chains. This, in
turn, leads to the fact that every system of DNA double helix should have an
individual, unique, conformational status in all blastomers. This would also
mean an individual methylation pattern.
References
1. Galimov E.M. Biological Fractionation of Isotopes. N.Y. etc.: Academic
Press Inc., 1985.
2. Ivanov A.A. Does the conformation of DNA depend on the differences in the
isotope composition of it’s threads? Russian Journal of Physical Chemistry.
B. 2007. vol. 2. 6: 649-652.
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ABSTRACT 88
EVIDENCE OF HYPOTHALAMIC DEGENERATION IN THE
ANX/ANX MOUSE
IAK Nilsson1, S Thams2, C Lindfors1, A Bergstrand2, S Cullheim2, T Hökfelt2,
and Jeanette E Johansen1
1
Department of Molecular Medicine and Surgery, Karolinska Institutet,
Stockholm, Sweden
2
Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden
The anx/anx mouse is an interesting model for studies concerning different
conditions of disturbed feeding behavior, such as anorexia nervosa, cachexia and
failure to thrive. Mice homozygous for the anorexia (anx) mutation are
characterized by poor feeding and die around 3-5 weeks after birth.
Histochemical studies of anx/anx mice have shown aberrant appearance of
transmitter and neuropeptidergic systems originating in the arcuate nucleus
(Arc) of the hypothalamus, important for the regulation of food intake and
energy metabolism. For example, by P21 a lower density of hypothalamic
appetite regulating neuropeptides, including Agouti gene-related protein
(AGRP) and neuropeptide y (NPY) has been detected in these mice. The
AGRP/NPY-system of the anx/anx mice develops normally until P12, when the
normal increase in fiber density ceases, in some areas even distinctly decreases.
This overlaps both in time and space with activation of microglia, indicating an
inflammatory and/or degenerative process. To further evaluate this process we
studied the expression of major histocompatibility complex (MHC) class Irelated molecules and markers for cellular reactivity in the hypothalamus of
anx/anx mice. MHC class I transcript and -related proteins were found in Arc,
both in neurons and glia. Using TUNEL labeling, a significantly higher number
of apoptotic cells were found in anx/anx hypothalamus compared to +/+ mice,
and active caspase 6 immunoreactivity was detected in degenerating NPY-fibers
as well as signs of ‘microglia-associated cell death’. In addition, Y1 receptorlabeled processes and soma of pro-opiomelanocortin (POMC) neurons, were
markedly decreased, but not until P21. These results support the hypothesis of
degeneration of hypothalamic arcuate neuron populations in the anx/anx mice,
where the AGRP/NPY system may be affected first and the changes in the
POMC system seem to be secondary.
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ABSTRACT 89
EVIDENCE OF HYPOTHALAMIC DEGENERATION IN THE
ANORECTIC ANX/ANX MOUSE
IAK Nilsson1, S Thams2, C Lindfors1, A Bergstrand2, S Cullheim2, T Hökfelt2,
and Jeanette E Johansen1
1
Stockholm, Sweden
2
The anx/anx mouse is an interesting model for studies concerning different
conditions of disturbed feeding behavior, such as anorexia nervosa, cachexia and
failure to thrive. Mice homozygous for the anorexia (anx) mutation are
characterized by poor feeding and die around 3-5 weeks after birth.
Histochemical studies of anx/anx mice have shown aberrant appearance of
transmitter and neuropeptidergic systems originating in the arcuate nucleus
(Arc) of the hypothalamus, important for the regulation of food intake and
energy metabolism. For example, by P21 a lower density of hypothalamic
appetite regulating neuropeptides, including Agouti gene-related protein
(AGRP) and neuropeptide y (NPY) has been detected in these mice. The
AGRP/NPY-system of the anx/anx mice develops normally until P12, when the
normal increase in fiber density ceases, in some areas even distinctly decreases.
This overlaps both in time and space with activation of microglia, indicating an
inflammatory and/or degenerative process. To further evaluate this process we
studied the expression of major histocompatibility complex (MHC) class Irelated molecules and markers for cellular reactivity in the hypothalamus of
anx/anx mice. MHC class I transcript and -related proteins were found in Arc,
both in neurons and glia. Using TUNEL labeling, a significantly higher number
of apoptotic cells were found in anx/anx hypothalamus compared to +/+ mice,
and active caspase 6 immunoreactivity was detected in degenerating NPY-fibers
as well as signs of ‘microglia-associated cell death’. In addition, Y1 receptorlabeled processes and soma of pro-opiomelanocortin (POMC) neurons, were
markedly decreased, but not until P21. These results support the hypothesis of
degeneration of hypothalamic arcuate neuron populations in the anx/anx mice,
where the AGRP/NPY system may be affected first and the changes in the
POMC system seem to be secondary.
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ABSTRACT 90
IDENTIFICATION OF A NOVEL SLC25 MEMBER THAT CAUSES
AUTOSOMAL RECESSIVE ATAXIA IN ENU-MUTAGENIZED MICE
A Karakostas, F Ioakeimidis, V Rinotas, G Kollias, and Eleni Douni
Institute of Immunology, Biomedical Sciences Research Center “Alexander
Fleming”, Vari, Greece
We have recently generated by random mutagenesis using the mutagen N-ethylN-nitrosourea (ENU), a novel mouse model of severe autosomal recessive
neurological disease characterized by ataxia, unsteady locomotion, episodic
crises, and growth retardation. The symptoms start at 3 weeks of age with severe
disease progression that leads to lethality of the majority of the mice within 7
weeks after birth. Using genome-wide linkage analysis we localized the
mutation in a 2.9Mb genomic interval containing 26 genes. Upon sequencing of
the candidate genes we identified a nonsense point mutation (C to T) in the
coding region of a novel gene member of the Solute Carrier Family 25 (SLC25)
that introduces a premature stop codon and results in a loss-of-function protein.
All SLC25 members are nuclear-coded proteins that are imported into the inner
mitochondrial membrane where they shuttle a variety of metabolites across it.
Until now, almost 50 SLC25 members have been identified whereas most of
them remain uncharacterized. Mutations in SLC25 genes impair mitochondria
functions and result in at least 10 various human diseases, by affecting either the
synthesis of ATP through oxidative phosphorylation, or the selective transport
of solutes in and out of the mitochondrial matrix. This novel SLC25 member is
highly conserved among various species, but its function remains completely
unknown. Our ongoing studies are focused on a) the identification of the
primary site of lesion in order to align the brain pathology of the mouse model
with that of similar human neurodegenerative diseases, b) the identification of
mitochondrial dysfunctions, and c) the analysis of the expression profile and the
function of this novel SLC25 protein which constitutes a novel pathogenic target
in neurological diseases such as ataxia.
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ABSTRACT 91
CONSOMIC ANALYSIS OF GENETIC FACTORS RELATED TO
TEMPORAL DIFFERENCE OF HOME-CAGE ACTIVITY BETWEEN
B6 AND MSM
Ayako Ishii1,2, Akinori Nishi1,2, Toshihiko Shiroishi1,3, Aki Takahashi1,2, and
Tsuyoshi Koide1,2
1
Mouse Genomics Resource Laboratory, NIG, 2 SOKENDAI, 3 Mammalian
Genetics Laboratory, NIG, Japan
We have been studying genetic basis of behavioral diversity using a variety of
mouse strains including wild-derived strains. Recent studies have shown that
genetic factors have no small effect on home-cage activity. The wild-derived
mouse strain MSM/Ms (MSM) exhibits higher activity in the home-cage than
C57BL/6 (B6), a commonly used laboratory strain. In addition, there is a clear
strain difference in the temporal pattern of within-day activity. We have
analyzed consomic strains derived from B6 and MSM to elucidate genetic
mechanism responsible for strain differences in the temporal regulation of
home-cage activity. The results showed that many genetic factors are related to
home-cage activity with a complex manner and different genetic factors related
to different temporal component of the activity. In order to identify a causative
gene related to different level of home-cage activity, we focused on one of the
loci on telomeric region of Chromosome 6. One of the sub-consomic strains
which has only a short segment (~5Mbp) of candidate locus from MSM
exhibited different activity suggesting existence of the causative gene in this
region. We are currently conducting comparative analyses of DNA sequences
and gene-expression levels of candidate genes using the sub-consomic strain and
B6 to find the causative gene in this region.
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ABSTRACT 92
GENETIC DETERMINANTS FOR INTRAMUSCULAR FAT CONTENT
AND WATER HOLDING CAPACITY IN MICE SELECTED FOR HIGH
MUSCLE MASS
Stefan Kärst and Gudrun A. Brockman
Humboldt Universität zu Berlin, Berlin, Germany
Intramuscular fat content and water holding capacity are important traits in
livestock as they influence meat quality, nutritive value of the muscle and
animal health. The intramuscular fat content may influence the animals'
metabolic stability via blood glucose levels and the taste of meat products,
whereas low water holding capacity leads to impaired meat processing
properties and does not meet consumers' requirements. This study was
performed to identify genomic regions affecting intramuscular fat content and
water holding capacity. As a model for livestock, we used the two inbred mouse
strains of the Berlin Muscle Mouse population. The inbred Berlin Muscle Mouse
strains 806 (BMMI806) and 816 (BMMI816) have been long term selected for
high muscle mass. Although both strains have increased lean mass, the strains
differ in intramuscular fat content. The intramuscular fat content of the
Musculus longissimus of BMMI806 mice is approximately 1.4 fold higher than
in BMMI816 mice. Additionally, lower water holding capacity was observed in
BMMI816 mice compared to BFMI806. In a reciprocal intercross between the
two strains, a F3 population with 345 mice was generated. All animals were
phenotyped and genotyped at 164 SNP markers. Linkage analyses revealed three
quantitative trait loci for intramuscular fat content on Chromosomes 7, 12 and
15 and two loci for water holding capacity on Chromosomes 2 and 17. These
loci explained 10% and 6% of the observed phenotypic variance in
intramuscular fat content and water holding capacity in the F3 population,
respectively. In part, the quantitative trait loci mapped for these traits in mice
coincide with homologue regions in pigs, in which quantitative trait loci for the
same traits were found. Therefore, the mouse strains and this F3 population are
interesting genetic resources to identify responsible genes, which could also
contribute to quantitative trait loci effects in other species.
129
ABSTRACT 93
DISCOVERING
NOVEL
REGULATORY
PATHWAYS
OF
IMFLAMMATION BY ENU MUTAGENESIS
Philip L Kong1, Emma Timms1, Andrew Coulson1, Kristina Blagoeva-Hubbard1,
Paul Potter1,2, and Marc Feldmann1
1
Kennedy Institute of Rheumatology, Imperial College London, United Kingdom
2
MRC Harwell, Harwell Science and Innovation Campus, Oxfordshire, United
Kingdom
Inflammation is a complex process whose outcome underpins many
physiological functions. Likewise, its dysregulation is the key driver in many
pathological conditions, including autoimmune diseases such as rheumatoid
arthritis. Over the years a multitude of regulatory mechanisms of inflammation
have been defined, including those driven by interleukin 10 (IL10) and
glucocorticoids. However despite intensive research significant gaps still exist in
our knowledge of their mechanisms of action, hampering our efforts to
understand the diseases in which these molecules play a part, and to design
therapies. In this screen of ENU-mutagenised mice, we aim to uncover novel
genes and pathways that are involved in the action of these anti-inflammatory
molecules. The phenotype we screen is the inhibition of cytokine production by
IL10 and dexamethasone in macrophages, a key cell type in effecting
inflammation in the joint. Bone marrow-derived macrophages are stimulated
with LPS to drive the production of pro-inflammatory cytokines, in the presence
or absence of a predetermined amount of IL10 or dexamethasone. Cytokine
production is determined by the multiplexing Luminex platform. Animals that
exhibit reduced responsiveness to IL10 or dexamethasone are selected for
follow-up studies using cells archived in liquid nitrogen. Some of the data from
the primary screen and the follow-ups are presented here. Pedigrees that harbor
significant and interesting phenotypes are then chosen for mapping studies in
order to identify the underlying mutation responsible for the phenotype.
130
ABSTRACT 94
EPIGENETIC CHANGES IN FRIEDREICH ATAXIA PATHOGENESIS
AND THERAPY
S Al-Mahdawi, C Sandi, R Mouro-Pinto, V Ezzatizadeh, and Mark A Pook
Brunel University, Uxbridge, UK
Friedreich ataxia (FRDA) is a lethal inherited neurodegenerative disorder caused
by a homozygous GAA repeat expansion mutation within intron 1 of the FXN
gene, which leads to reduced expression of the essential mitochondrial protein,
frataxin. Evidence indicates that the GAA mutation induces epigenetic changes
and heterochromatin formation, thereby impeding FXN gene transcription
elongation. In particular, studies using FRDA patient blood and lymphoblastoid
cell lines have detected increased DNA methylation of specific CpG sites
upstream of the GAA repeat, together with histone post-translational
modifications in regions flanking the GAA repeat. Our own studies have
revealed similar epigenetic changes in FRDA patient autopsy brain and heart
tissues, the primary systems that are affected in this disorder. Furthermore, we
have established GAA repeat expansion-containing human FXN YAC transgenic
mice that exhibit comparable epigenetic changes in brain and heart tissues to
those detected in FRDA patient tissues. We have thus developed a mouse model
that is a valuable resource for pre-clinical testing of epigenetic-acting
compounds that may target the FXN gene to increase frataxin expression. We
have established fibroblast and neural stem cell lines from our FRDA transgenic
mice, and several DNA methylation inhibitors, histone deacetylase inhibitors
and GAA-interacting compounds have been tested on these cultured cells,
resulting in the identification of good candidates to take forward to further preclinical and clinical studies.
131
ABSTRACT 95
A MOUSE MUTANT EXHIBITING A UNIQUE PATTERN OF
COCHLEAR INNER HAIR CELL DEGENERATION
Morag A. Lewis, Tracy J. Bussoli, Amy Taylor, Michael A. Cheong, Cordelia F.
Langford, and Karen P. Steel
Wellcome Trust Sanger Institute, Cambridge, UK
Deafness is the most common sensory deficit in the human population, but the
genetic basis for nonsyndromic hearing loss is still largely unknown. It is
thought that hundreds of genes may be required for development and function of
the ear, many of which remain undiscovered. Deaf mouse mutants are a
powerful tool for discovering genes which affect hearing and characterising the
pathways which, when perturbed, result in dysfunction of the ear. Bronx waltzer
is a spontaneous autosomal recessive mutant which has been previously
described. Homozygotes demonstrate rapid degeneration of vestibular hair cells
and cochlear inner hair cells at a late embryonic stage, resulting in deafness and
vestibular dysfunction, but outer hair cells remain intact. The mutation has been
mapped to a 2.7Mb region on Chromosome 5, but exon resequencing has failed
to find a potential causative mutation. We have investigated the mutation using
numerous methods including Illumina resequencing of the entire critical region,
analyses of which detected multiple mutations ranging from large deletions to
single nucleotide polymorphisms. We are currently studying several candidate
genes which are expressed in the organ of Corti. The pattern of degeneration of
bronx waltzer hair cells is unique, so characterisation of the gene responsible for
the phenotype will enhance understanding of the development of the ear and
potentially lead to more effective therapies for hearing loss.
132
ABSTRACT 96
A COMPREHENSIVE WHOLE-GENOME MAP OF ENDOGENOUS
RETROVIRAL ELEMENTS AND THEIR FUNCTIONAL EFFECTS
ACROSS 17 LABORATORY MOUSE STRAINS
Thomas M Keane1, K Wong1, J Flint2, W Frankel3, and David J Adams1
1
Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton,
Cambridge, United Kingdom
2
Wellcome Trust Centre for Human Genetics, Oxford University, Oxford,
Oxford, United Kingdom
3
The Jackson Laboratory, The Jackson Laboratory, Bar Harbor, ME, USA
It has been estimated that endogenous retroviral elements (ERVs) are a
significant source (~10%) of spontaneous germline mutations among laboratory
mouse strains. Two high-copy families of ERVs in particular, IAP and ETns,
have been found to be responsible for the vast majority of these mutations. As
part of the Mouse Genomes Project (http://www.sanger.ac.uk/mousegenomes),
we have recently completed deep paired-end illumina sequencing of 17 key
mouse laboratory strains to between 20-35x depth. In this study, we report the
first use of new sequencing technologies data to catalogue the full repertoire of
the major classes of ERVs (IAP, ETn, MLV, LINE, and SINE) across these
strains. Our strategy utilises paired-end information by finding clusters of matepairs where either only one end maps well to the reference genome and the other
to a set of ERV reference sequences or incorrectly mapped pairs where one end
maps to a canonical copy in the reference. From the initial set of 1-2kb call
regions, we then further refine the calls by using the read depth information to
identify the exact breakpoints. We assess the accuracy of our calls by comparing
the set of calls we make in the C57B6NJ strain sequenced by us compared to the
insertions annotated in the C57B6J reference genome. We find extremely high
concordance between these two sets with just a few differing insertions. We
have also compared our findings to a large set of manually PCR validated
insertions falling into introns across five strains and find that our illumina
predictions show extremely high confirmation rates. In our analysis, we have
focused on young ERVs that occur in just a single strain and compared this set
of ERVs found across the strains to existing expression data finding several
examples of gene expression being potentially affected by intronic or proximal
ERVs and use this information to infer potential phenotypic traits amongst the
strains.
133
ABSTRACT 97
PHENOTYPING OF KNOCKOUT MICE USING BACTERIAL
PATHOGENS AS PART OF THE WELLCOME TRUST SANGER
INSTITUTE’S MOUSE GENETICS PROGRAMME
Leanne Kane, Lynda Mottram, Simon Clare, Jacqui White, Ramiro Ramirez –
Solis, and Gordon Dougan
All the Mouse genome project team at the Sanger Institute
Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus,
Cambridgeshire, UK
The Wellcome Trust Sanger Institute’s Mouse Genetics Programme is running
an extensive sequence/phenotype driven mutagenesis programme as part of
international efforts aimed at mutating over 95 percent of known mouse genes in
embryonic stem cells. The knockout mice, resources and data generated in this
programme are then freely available to the scientific community. All mutant
mouse lines generated in this high throughput programme are examined using a
common battery of phenotyping tools. At the Sanger Institute a component of
this phenotyping programme involves challenging the knockout mice with
selected pathogens. We are currently using two standard models of infection Salmonella Typhimurium an intracellular pathogen which induces a systemic
disease in mice similar to that of human typhoid fever and Citrobacter
rodentium a natural mouse pathogen which forms attaching and effacing lesions
on the surface of the lumen to colonise the host gastrointestinal tract in a similar
way to enterohaemorrhagic E.coli (EPEC). We have screened over 250
targeted mouse knockout lines and have found 15 phenotypes ranging in
severity from subtle antibody differences to mice succumbing rapidly to the
infection.
134
ABSTRACT 98
IMPRINTED METHYLATION VS. IMPRINTED EXPRESSION -ARE
THEY NECESSARILY ASSOCIATED?
John D. Calaway1, Hyuna Yang2, Megan E. Hanson3, Ezequiel C. Cambranis3,
Maria A. Perez-Lamigueiro3, David L. Aylor1, Leonard McMillan1, Gary A.
Churchill2, Fernando Pardo-Manuel de Villena1, and Elena de la CasaEsperon3,4
1
University of North Carolina, Chapel Hill, NC, USA
2
3
University of Texas Arlington, Arlington, TX, USA
4
Regional Center for Biomedical Research. University of Castilla-La
Mancha/Albacete Science and Technology Park. Spain
Imprinted genes are preferentially expressed from either the maternal or the
paternal copy, and their pattern of expression has been correlated with the
methylation status of nearby CpG sites. At these sites, only one of the two
alleles is methylated and alterations of these differentially methylated regions
(DMRs) can disrupt imprinted expression. While currently known DMRs have
been identified due to their proximity to known imprinted genes, we have
adopted the opposite strategy: to search for novel DMRs in order to uncover
nearby imprinted genes. Our genome-wide survey of the mouse methylome has
allowed us to identify several DMRs. Most of them are associated to known
imprinted genes, but not all: such is the case of a DMR located in the 3' of the
Actn1 gene, which has never been tested for imprinting. Consequently, we have
analyzed the expression of this gene in heterozygous mice resulting from
reciprocal crosses, both male and female, in diverse tissues. In spite of finding
widespread differential methylation, none of the tissues studied show any
indication of imprinted expression. Our results suggest that imprinted expression
of Actn1 might be restricted to a specific cell type and/or developmental stage,
while imprinted methylation of Actn1 is maintained in most cells. Alternatively,
our observations may support our hypothesis that parental origin-dependent
differences in methylation have been selected in sexually reproducing organisms
for reasons other than gene expression regulation.
135
ABSTRACT 99
DUAL ACTIVITY ON A SINGLE LOCUS: EXISTENCE OF A NOVEL
RNA CONTROL MECHANISM FOR MICRORNA-650 AND
IMMUNOGLOBULIN LAMBDA LIGHT CHAIN VARIABLE GENES IN
PRIMATES
Sabyasachi Das, Jianxu Li, Masayuki Hirano, Christopher L. Haga, Murali
Gururajan, Götz R.A. Ehrhardt, and Max D. Cooper
Department of Pathology and Laboratory Medicine, Emory Vaccine Center,
School of Medicine, Emory University, Atlanta, GA, USA
MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate gene
expression post-transcriptionally in a wide spectrum of biological systems. A
survey of the genomic context of human microRNA (miRNA) genes reveals that
the miR-650 gene overlaps with the protein-coding region of the
immunoglobulin lambda light chain variable (IGVL) gene in the same
transcriptional orientation.
This genomic organization offers a unique
opportunity to explore the evolutionary and functional consequences of
overlapping miRNA and protein coding genes. The miR-650-bearing IGVL
genes are present in multiple copies and belong to a specific phylogenetic clan
(clan II). Sequence comparison and structural prediction indicate that the
accumulation of nucleotide changes in the leader exons of clan II IGVL genes
has led to the generation of functionally stable miRNA hairpins. In contrast to
the expectation that expression of miRNAs overlaps with other genes in the
same transcriptional orientation, our analysis of ten different cell lines
demonstrates that, using the same promoter, the miR-650 gene is transcribed
independently of the IGVL gene. This suggests the existence of a novel RNA
control mechanism by which the same locus is expressed as either IGVL (VJrearrangement dependent) or miRNA (VJ-rearrangement independent) in
different cells. Our study on ten mammalian genome sequences suggests that the
control mechanism for mutually exclusive expression of miR-650 and IGVL
genes evolved in the primate lineage.
136
ABSTRACT 100
IDENTIFICATION OF POSITIONAL CANDIDATES FOR CHR15
OBESITY QUANTITATIVE TRAIT LOCI USING MICROARRAY AND
RT-PCR ANALYSES OF F2 CONGENIC CROSSES IN MICE
K Cirnski1, M Stanonik1, R Keber1, P Juvan2, R Košir2, D Rozman2, and Simon
Horvat1,3
1
Biotechnical Faculty, Dept. Anim. Sci. University of Ljubljana, Domžale,
Slovenia
2
Institute of Biochemistry, Faculty of Medicine, University of Ljubljana,
Slovenia
3
National Institute of Chemistry, Ljubljana, Slovenia
Obesity is a risk factor for a number of chronic diseases including diabetes,
cardiovascular diseases and cancer and is controlled by a complex interplay
between genetic and environmental factors. Additional genes from the common
polygenic form of obesity need to be identified to elucidate the mechanisms of
susceptibility or resistance to obesity development. We previously identified a
Chr 15 quantitative trait locus (QTL) Fob3b in lines of mice selected on high
(Fat line) and low (Lean line) body fat content that represent a unique model of
polygenic obesity. Follow up studies revealed that Fob3b consists of at least two
separate linked QTLs Fob3b1 and Fob3b2. The objective of the study presented
here was to identify differentially expressed candidates for Fob3b1 and Fob3b2
using microarray and quantitative RT-PCR analyses. Brain, gonadal fat and liver
RNA of homozygotes for alternative alleles from an F2 cross between the
congenic line containing Fob3b1 and Fob3b2 QTL and Fat line was analyzed.
Affymetrix GeneChip 1.0 ST arrays were used and analyzed using modified ttest (R/Bioconductor/Limma) of individual genes, gene enrichment analysis
(R/Bioconductor/PGSEA) of gene sets from GO terms and KEGG pathways,
and Venn analysis of differentially expressed genes/gene sets. A focused
analysis on Chromosome 15 revealed 1 candidate in brain, 2 in gonadal fat and 9
in liver. Most of these genes were found to be involved in obesity-related
processes such as biosynthesis of fatty acids, steroid hormone metabolism,
glycerophospholipid metabolism, PPAR signaling pathway and Wnt signaling
pathway. A comprehensive quantitative RT-PCR analysis to validate the
microarray candidates in a larger set of F2 animals is under way and will be
presented. This study should enable us to narrow down the list of candidates for
the obesity Fob3b1 and Fob3b2 QTL loci. Identifying new obesity loci should
help to uncover important inherited risk factors and provide novel diagnostics
and therapeutic approaches.
137
ABSTRACT 101
CREZOO - THE EUROPEAN VIRTUAL REPOSITORY FOR CRE
DRIVER STRAINS
Christina Chandras1, Michael Zouberakis1, Damian Smedley2, Nadia
Rosenthal3, and Vassilis Aidinis1
1
B.S.R.C. Al. Fleming, Athens Greece
2
European Bioinformatics Institute, EMBL, Wellcome Trust Genome Campus,
Hinxton, Cambridge, UK
3
EMBL-Monterotondo Outstation, Italy
CreZOO (www.fleming.gr/crezoo) is the European database of genetically
modified mice expressing cre recombinase and other conditional systems under
the control of cell-specific promoters/enhancers. Its aim is to promote European
collaboration towards unified mouse databases and deposition of existing cre
mouse strains into EMMA and to share and publicize information on mouse
characteristics and availability. Transgenic mice carry detailed information on
promoter(s), alleles and genetic background (DNA origin, targeted, host and
backcrossing background), in addition to recombination efficiency, specificity,
integration site and copy number where available. Each promoter/gene index
includes IDs and direct links to MGI, Ensembl and NCBI’s Entrez Gene
database. The site of transgene expression is described using the Mouse adult
gross anatomy (MA) as developed by the Jackson Laboratories, while
embryonic development is standardised based on EMAP’s Mouse gross
anatomy and development ontology. Availability (e.g. live mice, cryopreserved
embryos, sperm, ES cells) is clearly indicated, along with handling and
genotyping details (in the form of documents or hyperlinks) and all relevant
contact information (including EMMA and Jax/IMSR hyperlinks where
available). CreZOO is the front-end of a normalized PostgreSQL database, built
around EJBs technology and is deployed on a Glassfish application server.
CreZOO also enables programmatic access via web services. Its design is based
on the users’ convenience and is thus an easy to use database, offering a userfriendly query interface and providing instant access to the list of transgenic
mice, transgenes and alleles. Finally, handling and genotyping instructions as
well as the primary and additional references are also available in the final tab
for each mouse. CreZOO is still under construction and is created in the context
of the CREATE consortium, a core of major European and international mouse
database holders and research groups involved in conditional mutagenesis,
primarily to develop a strategy for integration and dissemination of cre driver
strains for modeling aspects of complex human diseases in the mouse. CREATE
aims integrate new and existing information on cre driver strains, through a
virtual international repository of genetically modified mice expressing cre
recombinase, the cre8 portal (http://www.creline.org/search_cre_mice).
138
Participating databases (CreZOO/EU, MGI's Recombinase/USA and Cre-XMice/Canada) will exchange/contribute seven agreed datafields to the cre8
portal thus fulfilling the urgent need on the production and expansion of an
international creZOO to complement the conditional mouse alleles currently
being generated worldwide.
139
ABSTRACT 102
HOST
IMMUNE
RESPONSES
AND
GENETIC
FACTORS
MODULATING RESISTANCE TO SALMONELLA ENTERICA
SEROVAR TYPHIMURIUM IN THE INBRED MOUSE STRAIN
SPRET/EI: A ROLE FOR NEUTROPHILS
Lien Dejager, Iris Pinheiro, Pieter Bogaert, Liesbeth Huys, and Claude Libert
Molecular Mouse Genetics Unit, VIB and Ghent University, Ghent, Belgium
Infection with Salmonella enterica serovar typhimurium is a complex disease in
which the host-bacteria interactions are strongly influenced by genetic factors of
the host. We demonstrate that SPRET/Ei, an inbred strain derived from Mus
spretus, is resistant to S. typhimurium infections. The kinetics of bacterial
proliferation as well as histological examinations of tissue sections suggest that
SPRET/Ei mice can control bacterial multiplication and spreading despite
significant attenuation of the cytokine response. The resistance of SPRET/Ei
mice to S. typhimurium infection is associated with increased leukocyte counts
in circulation and enhanced neutrophil influx into the peritoneum during the
course of infection. A critical role of neutrophils was confirmed by neutrophil
depletion: neutropenic SPRET/Ei mice were sensitive to infection with S.
typhimurium and showed much higher bacterial loads. To identify genes that
modulate the natural resistance of SPRET/Ei mice to S. typhimurium infection,
we performed a genome-wide study using an interspecific backcross between
C3H/HeN and SPRET/Ei mice. This analysis demonstrates that at least two loci,
located on chromosomes 6 and 11, affect survival following lethal infection with
S. typhimurium. These two loci contain several interesting candidate genes,
which may have important implications for the search for genetic factors
controlling Salmonella infections in humans and for our understanding of the
complex host-pathogen interactions in general.
140
ABSTRACT 103
LPS-RESISTANCE OF THE MOUSE STRAIN SPRET/EI: AN
IMPORTANT ROLE OF THE X-CHROMOSOME
Iris Pinheiro, Lien Dejager, Tina Mahieu, Marnik Vuylsteke, and Claude Libert
Molecular Mouse Genetics Unit, VIB and Ghent University, Ghent, Belgium
Mus spretus diverged from the Mus musculus complex of house mouse
subspecies about 1.5 million years ago. Several inbred strains, as SPRET/Ei (S),
have been derived from this species. Recently, we have described that this strain
shows a remarkable resistance in models of acute inflammation, e.g. triggered
by cytokines or by bacterial components, as lipopolysaccharides (LPS).
Objectives: Study the genetic basis for X-located LPS-resistance observed in
SPRET/Ei mice. Methods: Linkage analysis, microarrays analysis, in vitro
models (bone marrow and ES-cells derived macrophages), in vivo models,
tetraploid aggregation. Results: The nature of S LPS-resistance is complex, and
multiple genetic factors seem to be involved. It is a dominant trait, and two
major protective loci, namely on chromosomes 2 and X and two minor
protective loci on chromosomes 10 and 13 were identified by linkage analysis.
Moreover, F1 females resulting from the cross between C57BL/6 (B) females
and S males, and therefore carrying the S X-chromosome, display higher
resistance than F1 males. Both in vivo and in vitro results indicate that induction
of IL6 after LPS challenge is lower in (BxS)F1 females. Furthermore, (SxB)F1
males resulting from the reverse cross, i.e. between S females and B males, do
also show an increased resistance to LPS when compared to (BxS)F1 males, a
fact confirming the contribution of the S X-chromosome to this trait. A genomewide expression profile on both parental strains and F1 offsprings of both sexes
was performed by using Affymetrix microarrays. A few X-located candidate
genes were found to be differentially regulated by LPS in F1 females and males
bone marrow derived macrophages. The contribution of these genes to LPSresistance of both F1 females and S mice will be further investigated by the use
of in vitro models as well as by the generation of (BxS)F1 male mutants by
tetraploid aggregation. Conclusion: The use of SPRET/Ei as a mouse model can
be of valuable use as a tool to disclose the molecular mechanism which is
responsible for the observed LPS-resistance. Ultimately we hope to contribute to
the development of a new therapeutic approach in the treatment of endotoxemia
and sepsis.
141
ABSTRACT 104
COMPLETE KNOCKOUT OF THE ADRENOCORTICAL DYSPLASIA
GENE ENCODING THE SHELTERIN PROTEIN TPP1 IS
ASSOCIATED WITH TELOMERE DYSFUNCTION AND EARLY
EMBRYONIC LETHALITY
Gail A Osawa1, C Harris1, T Kibe2, T de Lange2, S Kalantry1, and CE Keegan1
1
University of Michigan, Ann Arbor, MI. USA
2
Rockefeller University, New York, NY, USA
Adrenocortical dysplasia (Acdacd) is a spontaneous autosomal recessive mouse
mutation that originated on the DW/J strain. Homozygous acd mutant mice
exhibit a strain-dependent pleiotropic phenotype; on the DW/J strain, the
phenotype is lethal shortly following birth and includes caudal truncation,
vertebral segmentation, and limb patterning defects. In contrast, acd mutant
mice on a mixed DW/J X CAST/Ei background are growth retarded,
hyperpigmented, and infertile but can survive into adulthood. We previously
characterized the acd mutation as a splicing defect in the Acd gene, which
encodes TPP1, a component of the shelterin complex that functions to maintain
telomere integrity. Because of the critical role of Tpp1 in the shelterin complex,
we suspected that acd is a hypomorphic mutation. In addition, previous studies
of acd mutant MEF cells showed worsening of the cellular phenotype following
shRNA knockdown of Acd expression. To examine the phenotype of a
complete knockout, we created a null allele by deleting exons 3 through 8 of the
Acd gene.
Acdnull MEF cells exhibit evidence of significant telomere
dysfunction similar to double Pot1a/Pot1b knockout cells, including telomere
dysfunction-induced foci, chromosomal fusions, and endoreduplication. In
addition, Acdnull cells show loss of POT1 proteins from the telomeres. These
data indicate that the main function of TPP1 is to allow POT1A and POT1B to
protect chromosome ends. To examine the mutant phenotype in early stage
embryos, we performed timed pregnancies with heterozygous Acdnull mice, and
we found no homozygous mutant embryos as early as E8.5. We are currently
analyzing blastocyst stage embryos to determine whether homozygous mutants
are present at these early timepoints. These studies demonstrate that the acd
mutation is hypomorphic and that Acd/TPP1 function is critical for telomere
integrity and is required at very early stages of embryonic development.
142
ABSTRACT 105
THE FACEBASE CRE DRIVER PROJECT: CREATING NEW MOUSE
TOOL STRAINS FOR CLEFTING RESEARCH
Stephen A. Murray, Jocelyn Sharp, John Flaherty, Thomas Gridley, and Leah
Rae Donahue
Orofacial clefting is one of the most common birth defects in humans, affecting
approximately 1 in 700 live births. This frequency highlights the complexity of
craniofacial morphogenesis, which requires precise regulation of gene
expression changes, alterations in cell physiology and morphogenic movements.
The mouse has played an instrumental role in advancing our understanding of
the mechanisms that govern mid-face and palate development. Future progress,
however, will require an increasingly sophisticated set of genetic models and
tools. As part of the NIDCR-funded FaceBase Consortium, we will generate 15
Cre driver strains to facilitate orofacial clefting research in the mouse. These
driver strains will comprise both inducible and non-inducible approaches, and
include a fluorescent marker for simultaneous visualization of Cre expression
patterns. The individual driver promoters have been chosen to encompass both
early midface and primary/secondary palate development and fusion. These
tools are designed to complement existing lines, allowing users to define gene
function in new cell types and regions. A combination of approaches will be
employed to produce the lines, including BAC transgenic, knock-in and
targeting to the ROSA26 locus. In addition to detailed characterization of Cre
functionality in the midface and palate, we will use our existing comprehensive
characterization pipeline to ensure specificity and carefully define any nontarget activity. All of this data will be made available as part of the FaceBase
effort, and lines will be distributed through the JAX FaceBase Repository,
which includes both Cre lines and other mouse models and tools useful in
clefting research. Development and rapid distribution of these lines will greatly
enhance the genetic “toolbox” available to the scientific community for
understanding the mechanisms that govern orofacial clefting.
143
ABSTRACT 106
EPIGENOME SEQUENCING COMES OF AGE IN DEVELOPMENT,
DIFFERENTIATION AND IMMUNE REGULATION MECHANISMS
OF MAMMALIAN RESEARCH
Ning Li and Jun Wang
Beijing Genomics Institute, Shenzhen, China
Epigenetics is a new field, defined as heritable changes in gene expression that
are not accompanied by changes in DNA sequence, which could open up many
possibilities for future development process control and disease treatment. DNA
methylation plays a key role in epigenome. There are numerous approaches to
decipher a whole genome DNA methylation profile (“methylome”), each
varying in cost, throughput and resolution. Bisulfite-sequencing is the golden
standard of these methods. Compared with it, methylated DNA
immunoprecipitation (MeDIP-Seq) and methyl-binding protein (MBD-Seq) are
complementary strategies. MeDIP-Seq is more sensitive to highly methylated,
high-CpG densities and MBD-Seq is more sensitive to highly methylated,
moderate-CpG densities. Bisulfite-Seq has high reliability and accuracy to
determine each cytosine methylation state. In our study, we have generated a
single-base-resolution human DNA methylation profile in peripheral blood
mononuclear cells (PBMC), using Bisulfite-Seq, which was determined by 20X
coverage genome-wide resequencing after bisulfite treatment. In this research,
essentially complete (92.62%) methylome of PBMC was reported, and only
<0.2% of non-CpG sites were methylated, demonstrating that non-CpG cytosine
methylation is negligible in human PBMC. Integration of this methylome data
with the previously determined genome sequence of the same Asian individual
analysed here, enabled a first assessment of allele-specific methylation (ASM)
differences between the two haploid methylomes of any individual. We estimate
that about 30% of the haploid differentially methylated regions (hDMR) may be
attributed to imprinting and about 70% to other mechanisms such as sequencedependent ASM, suggesting that imprinting may be more common than
previously thought. The study performed herein not only provides a
comprehensive method for detailed understanding of epigenetic regulation
mechanisms, but also serves as a catalyst for future studies of the epigenetic
mechanisms that regulates development, differentiation and immune regulation
mechanisms of mammalian research.
144
ABSTRACT 107
GENERATION
OF
CUSTOMIZED
AND
READ-TO-USE
GENETICALLY ENGINEERED MICE AT THE INSTITUT CLINIQUE
DE LA SOURIS – ICS (THE FRENCH MOUSE CLINICAL INSTITUTE)
Marie-Christine Birling, Andrée Dierich, Karim Essabri, Lydie Venteo,
Guillaume Pavlovic, Tania Sorg, and Yann Hérault
Institut Clinique de la Souris (ICS) and IGBMC, Illkirch, France,
http://www.ics-mci.fr/
The Institut Clinique de la Souris - ICS is a research infrastructure that provides
extensive services ranging from the development of mouse models to
comprehensive phenotyping. The Mutagenesis, Transgenesis and Molecular
Phenotyping Department is dedicated to the development and molecular
validation of new mouse models.
Four main services are offered through the Department:
1) The Genetic Engineering service provides expert advises, proposes
strategies and takes care of the design of new targeting constructs. All kind
of constructs can be developed in the service:
a. Constructs for random insertion of a transgene (traditional or BAC
construct),
b. Constructs targeted insertion of a transgene, or for targeted transgenesis
(Constitutive knock out, Tissue specific knock out, Point mutation,
Humanization, Flex models, etc)
The Genetic Engineering service is also in charge of the screening and
validation of the ES cell clones. Long range PCR analysis, sequencing of the
inserted fragment, as well as Southern blot analysis on the genomic DNA of
embryonic stem (ES) cells are routinely performed.
2) The ES cell service is dedicated to the electroporations, selections, picking,
amplification and karyotyping of the ES cell clones. New ES cell lines from
various genetic backgrounds have been derived. Notably, since 2009, the
standard electroporations are performed in an in house derived C57Bl6/N ES
cell line. This line has been shown to be quite efficient for homologous
recombination and germ line transmission.
3) The Genotyping service confirms the insertion of the transgene when the
mice are born. A PCR genotyping strategy is provided to the client at the
delivery of the model.
4) The Molecular Phenotyping service analyzes, and most often validate, the
functionality of the genetic modifications introduced in vivo. The techniques
choosen for these analyzes are dependent on the mouse model (qPCR, RTqPCR, Southern blot, Western blots, in situ Hybridization). The relevance of this
molecular analysis before the generation of phenotyping cohorts is very high.
145
In addition to the service activities of the department, internal research and
development programs (eg. development of new mouse tools) in concertation
with European funded project such as the Create consortium
(http://www.creline.org/) are carried out. Two main resources as freely available
the community:
-The CreERT2 zoo. More than 50 cell or tissue specific promoter driven
CreERT2 transgenic lines are available to the whole scientific community
(http://www.ics-mci.fr/crezoo.html). Most of these lines have been partially
validated and show at least specificity in the mRNA expression.
-The NR zoo: most of the nuclear receptors and their co-factors have been
developed as mouse conditional KO models (http://www.ics-mci.fr/nrzoo.html)
146
ABSTRACT 108
PUDDING - A NOVEL MODEL OF GLOMERULARNEPHRITIS
Paul K Potter1, L Wisby1, FW Tam2, S Wells1, T Hough1, HT Cook2, M
Cheeseman1, and SDM Brown1
1
MRC Harwell, Harwell Science and Innovation Campus, Oxfordshire, UK
2
Renal Unit, Division of Medicine, Hammersmith Campus, Imperial College,
London, UK
As part of the ongoing phenotype driven ENU mutagenesis programme at MRC
Harwell we identified mice from a recessive pedigree that exhibited normal
post-natal growth and behavior but at around 90 days of age rapidly
deteriorated, exhibiting clear signs of a debilitating disease. Further breeding
was carried out confirming this was an inherited recessive trait. Initial
histological analysis of kidneys in affected individuals indicated the mice had a
glomerulopathy. To further characterize renal function in these mice we carried
out clinical chemistry analysis of serum and urine. The mice developed
proteinuria and azotemia (serum creatinine 50-112 umol/L vs 12.8-13.5 umol/L
and urea >32 mmol/L vs 9 mmol/L in affected and aged matched controls
respectively). This is associated with reduced serum albumin concentration and
raised amylase. Further detailed histological analysis showed that these mice
had crescentic glomerulonephritis. The disease results in the rapid deterioration
of renal function around 90 days of age with only minimal changes in serum
creatinine levels at 50 days of age. Preliminary mapping studies indicate that
the causative mutation lies within a region spanning 80.1-93.2 Mb on
chromosome 1. These initial studies indicate that this is a novel model of
spontaneous glomerulonephritis and we are carrying out further investigations to
identify the underlying mutation and further define the pathogenesis of disease
in this line.
147
ABSTRACT 109
THE HARWELL AGEING SCREEN
Paul K Potter
MRC Harwell, Harwell Science and Innovation Site, Oxfordshire, UK
Diseases associated with ageing pose an increasing social and financial burden
on society and represent a vital imperative for research in the biomedical
sciences.
Despite the complications of genetic background in human
populations and the confounds of environment there has been considerable
advances, particularly through Genome-wide association studies (GWAS) in
identifying loci involved in diseases of aging. Nevertheless, this is only a first
step towards a more fundamental understanding of the genetic pathways
involved. Animal models are required both to test our understanding of these
pathways, as well as to provide the tools for developing and assessing
therapeutics. We are undertaking the first large-scale project to employ
mutagenesis and phenotyping programmes to specifically generate new
recessive models of late onset or age-related disease. The emphasis will be on
the exploration of phenotype space in aging mouse mutant populations
providing us with the opportunity to: identify genes and pathways involved in
age related disease, scrutinise these models for biomarkers of age related
disease, and provide better platforms for pre-clinical assessment of new
therapies for such diseases. Pedigrees will be aged up to 18 months und undergo
phenotyping across a wide range of disease areas. Analysis will occur at several
defined time points throughout the life of the mice and the phenotypes included
in the screening include diabetes and metabolism, neurobehaviour, cognitive
tests, bone analysis, renal function, cardiac disease, liver function, sensoneural
(vision and hearing), and a comprehensive clinical chemistry screening. Other
screens are also in development. Detailed histological analysis will be carried
out as part of the terminal analysis and a biobank of samples taken at different
ages will also be established for retrospective analysis. The age challenged mice
will be an important resource for many research groups, identifying novel genes
and pathways resulting in age-related phenotypes along with the potential to
discover new biomarkers.
148
ABSTRACT 110
GELSOLIN PLAYS A ROLE IN THE ACTIN POLYMERISATION
COMPLEX OF HAIR CELL STEREOCILIA
Philomena Mburu1, MR Romero1, Helen Hilton1, Andrew Parker1, Stuart
Townsend1, Yoshiaki Kikkawa2, and SDM Brown1
1
Medical Research Council Mammalian Genetics Unit, Harwell Science and
Innovation Campus, Oxfordshire, UK
2
Department of Bioproduction, Tokyo University of Agriculture, Yasaka,
Abashiri, Hokkaido, Japan
A complex of proteins scaffolded by the PDZ protein, whirlin, reside at the
stereocilia tip and are critical for stereocilia development and elongation. We
have shown that in outer hair cells (OHCs) whirlin is part of a larger complex
involving the MAGUK protein, p55, and protein 4.1R. Whirlin interacts with
p55 which is expressed exclusively in outer hair cells (OHC) in both the long
stereocilia that make up the stereocilia bundle proper as well as surrounding
shorter microvilli that will eventually regress. In erythrocytes, p55 forms a
tripartite complex with protein 4.1R and glycophorin C promoting the assembly
of actin filaments and the interaction of whirlin with p55 indicates that it plays a
similar role in OHC stereocilia. However, the components directly involved in
actin filament regulation in stereocilia are unknown. We have investigated
additional components of the whirlin interactome by identifying interacting
partners to p55. We show that the actin capping and severing protein, gelsolin, is
a part of the whirlin complex. Gelsolin is detected in OHC where it localizes to
the the tips of the shorter rows but not to the longest row of stereocilia and the
pattern of localisation at the apical hair cell surface is strikingly similar to p55.
Like p55, gelsolin is ablated in the whirler and shaker2 mutants. Moreover, in a
gelsolin mutant, stereocilia in the apex of the cochlea become long and straggly
indicating defects in the regulation of stereocilia elongation. The identification
of gelsolin provides for the first time a link between the whirlin scaffolding
protein complex involved in stereocilia elongation and a known actin regulatory
molecule.
149
ABSTRACT 111
NEW STERILE MODEL MOUSE FROM RIKEN ENU-BASED GENEDRIVEN MUTAGENESIS
Takuya Murata, Shigeru Makino, Ryutaro Fukumura, and Yoichi Gondo
RIKEN BRC, Ibaraki, Japan
RIKEN ENU-based gene-driven mutagenesis, “a reverse genetics in the next
generation”, is based on the two key resources; mutant genome DNA and frozen
sperm of thousands of ENU-mutagenized G1 male. By high-throughput
screening of mutant genomic DNA library, we can obtain allelic series of
mutations on the target gene. Despite of randomly induced mutations, we can
usually discover many missense mutations together with “null” mutation(s).
Compared with the knockout method, it is efficient to find multiple mutations
even in a cost effective way. The targeted mice are recovered by IVF technology
from the frozen sperm. Since thousands of point mutations were introduced in
all over the genome of each G1 mouse line, they are usually removed by
continuous backcrosses at least six generations. Along the way of the
backcrosses, the excess hetrozygotes at every generation may be utilized to the
intercrosses in order to pre-examine the “phenotypes even still in progress.” This
distinctive infrastructure has been widely opened to the research community
since 2002 (http://www.brc.riken.jp/lab/mutants/genedriven.htm). WNT/β −
catenin signaling is essential for various stages of development, including cell
fate determination, cell proliferation (tumorigenesis), maintenance of the stem
cell state, and so on. In the targeted analyses of the C429S mutant mouse line on
the β−catenin gene, we found unique phenotypes observed in the homozygotes
obtained from 12th backcrossed parents so far. The mutant homozygotes were
born normally escaping from the embryonic lethality; however, most of them are
sterile, despite oogenesis or spermatogenesis looked normal. In female, most
homozygotes showed hydrometrocolpos (uterine edema) resulting from vaginal
atresia (closure of vagina). In homozygous male, regardless of making vaginal
plug, seminiferous seemed to be affected, because many sperm cells were found
ectopically in seminal vesicle as well as its extra lobes. From the observation
along the seminal vesicle formation, these abnormal lobes were originated from
the caudal ends of the Wolffian duct, which normally develops to caudal vas
deferens, namely that the cell fate change occurred in the caudal Wolffian duct
of the homozygous male. The sterility, coinciding with persistent malformations
of the internal genitalia, has been previously reported in various human cases,
such as hydrometrocolpos (equivalent to our phenotype), PMDS (Persistent
Mullerian Duct Syndrome), MKKS (McKusick-Kaufman Syndrome), and so on.
However, the cases corresponding to our homozygous males have never
reported in both mouse and human, suggesting a new sterile model in this field.
150
ABSTRACT 112
GENETIC DIVERSITY AMONG C57BL/6 SUBSTRAINS BASED ON
SNPS
Kazuyuki Mekada1, Ikuo Miura1, Takehide Murata1, Atsushi Toyoda2, Mayu
Hirose1, Shigeharu Wakana1, Kuniya Abe1, and Atsushi Yoshiki1
1
RIKEN BioResource Center, Japan
2
National Institute of Genetics, Japan
The C57BL/6 mouse strain is the best-known inbred strain and is commonly
used to create congenic strains as the genetic background for spontaneous and
induced mutations. Since the 1950s, C57BL/6 has diverged to several substrains
including C57BL/6J and C57BL/6N substrains. Currently, over 20 substrains
have been established worldwide, and their phenotypic and genetic differences
have been reported. Large-scale mutagenesis programs to mutate all of the
protein-encoding genes in the mouse using gene trapping and targeting in
embryonic stem cells derived from C57BL/6N are ongoing. Therefore, it is
required for the correct interpretation of the data from the functional analyses of
these genes to clarify the genetic properties of the C57BL/6N substrains and the
differences between C57BL/6N and C57BL/6J. We have already reported over
30 single nucleotide polymorphisms (SNPs) in several C57BL/6 substrains.
Nevertheless, we have not yet recovered any SNP markers between C57BL/6J
and C57BL/6N on chromosomes 1, 12 and Y. Besides, no SNP markers that
can distinguish the C57BL/6N substrains have been identified. Recently, a more
detailed genome sequence information of the C57BL/6N is available from the
end sequencing program of C57BL/6N BAC clones by RIKEN BRC and the
National Institute of Genetics with support of NBRP, MEXT Japan, and the
resequencing project by the Wellcome Trust Sanger Institute. In this study, we
report recovery of new SNPs between the C57BL/6J and C57BL/6N substrains
using these information, and the genetic diversity among C57BL/6 substrains.
151
ABSTRACT 113
NOVEL ENU SCREENS FOR IMMUNE-RELATED PHENOTYPES
Vera M Ripoll1, Marie Hutchison2, Paul Potter2, and Roger Cox1
1
Mammalian Genetics Unit, Medical Research Council, Harwell, Oxfordshire,
UK
2
Mary Lyon Centre, Medical Research Council, Harwell, Oxfordshire, UK
The identification of novel alleles through ENU mutagenesis has provided clues
to a number of genes responsible for the immune response, contributing
substantially to our understanding of host defence, allergy and autoimmunity.
We are undertaking two different ENU screens to expand our library of
immune-related alleles and generate new models for inflammatory diseases. Our
phenotype-driven screen aims to identify ENU induced mutations that result in
abnormal blood leukocyte counts and cell ratios. To date we have screened 88
pedigrees (1700 animals) utilizing both haematology and Flow Cytometry
analyzers. We have identified at least 17 mouse lines with abnormal monocyte,
neutrophils and/or lymphocytes numbers. Five different pedigrees with
distinctive blood phenotypes were selected for inheritance testing. We are
currently undertaking further phenotypic analysis of these pedigrees as well as
carrying out the mapping of the mutated chromosomal loci to identify potential
gene candidates. We will describe up to date progress in these experiments. In a
gene-driven approach, we have screened a DNA archive of 10000 samples from
F1 ENU-mutagenized mice for novel mutations in macrophage-inflammatory
genes. One of our genes of interest known as Mpeg1 (macrophage-expressed
gene 1) is a macrophage-specific gene, involved in macrophage activation and
responses to bacterial and viral infection. A mutant with a stop codon, which
disrupts the most important protein domain, was re-derived. We have been
carrying out primary immune-phenotyping experiments in Mpeg1 heterozygous
and homozygous mutants. Preliminary data demonstrate that macrophages from
Mpeg1 homozygous mice are unable to uptake bacteria and exhibit impaired
phagocytosis rates. Mpeg1 homozygous macrophages secrete significantly
higher levels of nitric oxide than Mpeg1 heterozygous and wild type
macrophages. These results confirm the involvement of Mpeg1 in the endocytic
pathways and suggest a role for Mpeg1 in the macrophage antibacterial
responses.
152
ABSTRACT 114
GRB10 MODIFIES MPNST TUMORIGENESIS IN A MOUSE MODEL
OF NF1
Karlyne M. Reilly, Jessica Van Schaick, Keiko Akagi, Christina DiFabio. Robert
Tuskan, Sandra Burkett, and Jessica Walrath
Mouse Cancer Genetics Program, National Cancer Institute, Frederick, MD,
USA
As human GWAS studies make the identification of susceptibility genes for
cancer more accessible, mouse models of cancer provide a unique opportunity to
identify modifier genes for rare human cancers and modifiers that show complex
epistatic interactions. Neurofibromatosis type 1 (NF1) is one of the most
common genetics diseases affecting the nervous system, with patients
developing cancers of both the central and peripheral nervous systems. Although
these tumors are less common, they have very poor prognosis, and so a better
understanding of the biology underlying nervous system tumors is needed to
develop new therapeutic options. We are using a mouse model of NF1 in which
the Nf1 and Trp53 gene are mutated on the same chromosome in cis (NPcis
mice). These mice develop central and peripheral nervous system tumors
characteristic of NF1 and can be used to identify modifier genes of these tumors.
We have identified both strain-specific modifier effects and parent-of-originspecific modifier effects that show interesting patterns of interaction. We report
here the identification of an imprinted modifier gene, Grb10, on mouse
Chromosome 11. Grb10 is expressed at different levels in tumors from progeny
of NPcis mutant mothers compared to tumors from progeny of NPcis mutant
fathers and correlates with differences in tumor susceptibility. We show that
mutation of Grb10 in the cis configuration with Nf1 and Trp53 alters
susceptibility to peripheral nerve tumors in vivo.
153
ABSTRACT 115
HOMOLOGY MAPPING: DEVELOPMENT OF THE MOUSE
HINDLIMB MUSCULOSKELETAL MODEL USING THE HUMAN
MUSCULOSKELETAL MODEL AND SCLERAXIS (SCX)-GFP MOUSE
Satoshi Oota1, Nobunori Kakusho2, Yosuke Ikegami3, Kazuyuki Mekada1, Koh
Ayusawa3, Hirotaka Imagawa3, Yuichi Obata1, Ryutaro Himeno4, Hideo
Yokota2, Yoshihiko Nakamura3, and Atsushi Yoshiki1
1
BioResource center, RIKEN, Tsukuba, Japan
2
Advanced Science Institute, RIKEN, Wako, Japan
3
Department of Mechano-Infomatics, The University of Tokyo, Tokyo, Japan
4
Advanced Center for Computing and Communication, RIKEN, Wako, Japan
Recently, the hypothesis-driven strategy has become useful for analyzing
movement disorders of laboratory mice. This new strategy possesses a
complementary role for the traditional comprehensive battery strategy. In order
to acquire detailed data on motor function required by this new strategy,
biomechanics is an ideal framework. However, difficulties in the direct
application of biomechanics to laboratory mice remain. One burdensome
difficulty is the development of a musculoskeletal model that can be used to
perform biomechanical analyses on subjects. Because of the heavy requirements
in the medical and athletic areas, detailed human musculoskeletal models have
been elaborated. On the other hand, no fine-grained musculoskeletal model of
the laboratory mouse has been developed to date. Unlike humans, knowledge of
the functional anatomy of the mouse is limited. Although MRI scan is a
conventional methodology for obtaining data on functional anatomy, its spatial
resolution is insufficient for mice due to their considerably small sizes. To
overcome this problem, we developed two alternative methods:
(1)Homology mapping. The human and mouse skeletal systems are
evolutionarily conserved. Theoretically, it is possible to map in silico part of the
human muscular system onto an existing mouse skeletal model according to
evolutionary relationships. We developed an algorithm to homologically map
the human muscular system on the mouse skeleton.
(2)Scanning transgenic mice. Using transgenic mice (the limb tendon-specific
Scleraxis (Scx)-GFP reporter line), we obtained 3D geometries of limb tendons
using a 3-dimensional internal structure microscope. We estimated
musculotendon geometries with regard to skeletal elements by integrating them
with X-ray CT scan data.
These methods are not only useful for developing a musculoskeletal model but
also provide insights on evolutionary difference between humans and mice.
154
ABSTRACT 116
MUTATION IDENTIFICATION IN MICE BY EXOME SEQUENCING
Laura Reinholdt1, David Bergstrom1, Steve Murray1, Michelle Curtain1, Carol Bult1,
Joel Richardson1, Lucy Rowe1, Mary Barter1, Daniel J. Gerhardt2, Mark D'Ascenzo2,
Todd Richmond2, Tom J. Albert2, Jeffrey A. Jeddeloh2, Benjamin Kile4, Ivo Gut5, Jay
Shendure3, and Leah Rae Donahue1
1
2
Roche NimbleGen, Inc., Madison, WI, USA
3
University of Washington, Department of Genome Sciences, Seattle, WA USA
4
Walter and Eliza Hall Institute of Medical Research, Parkville Victoria, Australia
5
Centre National de Génotypage, Evry, France
Traditionally, mutations in the mouse are identified via genetic mapping, a process
that can take several years and requires ample vivarium space. However, with the
advent of high throughput sequencing technology, mutations can be discovered with
minimal mapping data. Potential high throughput sequencing approaches to
mutation discovery include whole genome sequencing or targeted re-sequencing via
capture technologies. Currently, the sequencing and analysis of whole genomes
remains burdensome and expensive for many laboratories. Targeted re-sequencing
is less expensive and the data are compensatingly more manageable, but this
technique requires substantial genetic mapping and the design and purchase of
custom capture tools (i.e. arrays or probe pools). Targeted re-sequencing of the
coding portion of the genome, i.e. the ‘exome’, provides an opportunity to resequence mouse mutants with minimal mapping data and alleviates the need for a
custom array / probe pool for each mutant. This is critical for large sets of mice,
such as those in The Jackson Laboratory’s Mouse Mutant Resource (MMR); a
program, which has been funded to provide spontaneous and induced mutant mouse
models for 50 years. To significantly reduce the time, effort and cost of mutation
detection, we have collaborated to develop a sequence capture probe pool
representing the mouse exome. This exome is a collection of 203,225 exonic
regions comprising just over 54.3 Mb of target sequence, and is based on a unified
gene set. The unique design consists of non-redundant gene predictions from NCBI,
Ensembl and Vega. Our preliminary data show that capture sensitivity is high,
>96.7% of the targeted bases are covered with just one lane of 75 bp paired end on
the Illumina GAIIx (per 4 Gbp of mappable mate-pairs). The capture specificity is
also high, >75% reads from the lane come from the targeted bases. The
consequence of this high efficacy is that the platform provides sufficient coverage to
detect heterozygosity. We have found the genetic background to only modestly
impact the performance of a capture probe pool based upon the C57BL6(mm9)
reference. Lastly, and most importantly, we have successfully used this approach to
identify multiple spontaneous and induced mutations in the mouse.
155
ABSTRACT 117
STATUS OF THE US LINES OF THE COLLABORATIVE CROSS:
COMPLETED LINES
Darla R Miller1, Ryan J Buus1, Jennifer N Shockley1, Stephanie D Hansen1,
Ginger D Shaw1, Terry J Gooch1, Jason S Spence1, Timothy A Bell1, Catherine E
Welsh2, Leonard McMillan2, Wei Wang2, Jeremy Wang2, Eric Yi Liu2, Kenneth F
Manly3, Gary A Churchill4, David W Threadgill5, and Fernando Pardo-Manuel
de Villena1
1
Center for Genome Science
2
Department of Computer Science; University of North Carolina Chapel Hill,
NC, USA
3
Department of Biostatistics, University at Buffalo, Buffalo NY, USA
4
5
Department of Genetics, North Carolina State University, Raleigh, NC, USA
The Collaborative Cross (CC) is a large panel of recently established multiparental recombinant inbred (RI) mouse lines specifically designed to overcome
the limitations of existing mouse genetic resources for analysis of phenotypes
caused by combinatorial allele effects. The CC is the only existing mammalian
resource that has high and uniform genome-wide variation effectively
randomized across a large, heterogeneous, and infinitely reproducible population
in order to support data integration across environmental and biological
perturbations, across space (different labs) and time (temporal studies). Since
the last status report published in 2008 there have been significant changes in
the production of the US arm of the CC, including the relocation of the CC to
UNC, the constitution of an External Advisory Board, the naming scheme for
completed CC lines and the establishment of an MTA to facilitate access,
distribution and preservation of CC lines from the different arms. There have
been changes in the breeding scheme in later generations of inbreeding, the
development of a medium density SNP panel optimized for the CC and the use
of this panel to accelerate inbreeding through selective mating based on marker
genotypes. We will report on the completion of the first CC line, the status of
the lines undergoing marker assisted inbreeding and access to CC mice, highdensity genotypes and haplotype reconstructions of CC related resources
(http://csbio.unc.edu/CCstatus/).
156
ABSTRACT 118
WHOLE GENOME RESEQUENCING OF ESTONIAN DAIRY COW
(ESTONIAN HOLSTEIN)
Rutt Lilleoja1,2, Ü Jaakma1, and S Kõks1,2
1
Estonian University of Life Sciences, Tartu, Estonia
2
University of Tartu, Tartu, Estonia
The bovine reference genome currently available from public databases is one of
a Hereford female (Btau_4.0). We present here whole genome resequencing of
Estonian dairy cow using next generation sequencing. Whole genome sequence
of a female Estonian Holstein was generated using massively parallel
sequencing technology. 700 million short sequencing reads were obtained from
50-bp mate-paired library with a mean insert size of 1500 bp. All data was
aligned against Btau_4.0 Hereford reference sequence using Bioscope v1.2. We
were able to map over 75% of the reads to the reference, that is 42.3 gigabases
of sequence resulting in an avarage of 16-fold coverage. Further analysis of our
data is ongoing and the results will be presented during the conference meeting.
157
ABSTRACT 119
HYPOTHALAMIC MITOCHONDRIAL DYSFUNCTION, A POSSIBLE
CAUSE OF ANOREXIA
Charlotte E Lindfors1#, Ida A Nilsson1,2#*, Pablo M Garcia-Roves3, Aamir R
Zuberi4,5, Mohsen Karimi6, Lea R Donahue4, Derry C Roopenian4, Jan Mulder7,
Mathias Uhlén8, Tomas J Ekström6, Muriel T Davisson4, Tomas Hökfelt2, Martin
Schalling1, and Jeanette E Johansen1
1
Stockholm, Sweden
2
3
Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm,
Sweden
4
The Jackson laboratory, Bar Harbor, Maine, USA
5
Present address: Pennington Biomedical Research Center, Louisiana State
University, Baton Rouge, LA, USA
6
Department of Clinical Neuroscience, Karolinska Institutet Stockholm, Sweden
7
European Neuroscience Institute at Aberdeen, University of Aberdeen,
Aberdeen, UK
8
Department of Biotechnology, AlbaNova University Center, Stockholm, Sweden
#equal contribution
Human dysfunction of the oxidative phosphorylation system (OXPHOS) and
complex I (CI) can be associated with poor feeding and failure to thrive (FTT).
These phenotypic traits are also seen in the anx/anx mouse which exhibits
disturbed feeding behavior and aberrances, including signs of
neurodegeneration, in peptidergic neurons and neurotransmitter systems
important for the regulation of food intake in the hypothalamic arcuate nucleus
(Arc). In rodents, the Arc neuronal networks develop primarily during the first
three postnatal weeks, a very energy demanding period. We therefore
hypothesized that the starvation and degenerative phenotypes in the anx/anx
mouse could be related to defects in the OXPHOS and possibly CI deficiency.
Via an Affymetrix expression analysis on Arc and mapping of the anx locus, we
identified one of the CI assembly factors, the Ndufaf1 gene, as an excellent anx
candidate gene. By Taqman realtime-PCR and western blots we showed that the
Ndufaf1 gene and protein is approximately 50% downregulated in anx/anx mice
and we also showed that it is specific to the anx-allele. Furthermore, native
Western blots demonstrate decreased hypothalamic levels of fully assembled CI,
and increased levels of partly assembled CI in anx/anx mice. Finally, we show
evidence for oxidative stress and reduced efficiency of CI in anx/anx
hypothalamus. These and previous results provide evidence that the anorexia
and FTT of the anx/anx mouse are caused by hypothalamic neurodegeneration
following dysfunction of mitochondrial CI respiration.
158
ABSTRACT 120
HIGH-THROUGHPUT GENOTYPING AND QUALITY CONTROL OF
GENE-TARGETED MUTATIONS IN THE MOUSE
Edward Ryder, Diane Gleeson, Debarati Bhattacharjee, Ross Cook and the
Mouse Genetics Programme
Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton,
UK
The Sanger Institute is an active member of the international KOMP and
EUCOMM consortiums, whose goal is to produce conditional knockouts in ES
cell lines and mice as a resource for the research community. As part of these
projects and the EUMODIC European phenotyping collaboration, the Mouse
Genetics Programme (MGP) is involved in the generation of mouse mutants
from the ES cell stage to primary phenotyped mice. To establish a mutant
colony progeny from chimeras must be initially genotyped to determine whether
they carry the mutation in the germ line, and in subsequent generations to see if
they are wild type, heterozygous or homozygous. Tissue samples are genotyped
by a combination of Neo count qPCR and short range PCR, which allows us to
process up to 10,000 samples per week. Once a sufficient number of mice have
been generated they are sent as cohorts to the phenotyping pipelines for further
study and analysis, returning to genotyping for verification at necropsy. QC
methods are discussed for confirming the targeting and mutant allele structure
prior to injection, and in mouse colonies prior to release to the phenotyping
pipelines, external laboratories and the EMMA and KOMP repositories. This is
in conjunction with the newly established EUCOMM minimum genotyping
standard for mice generated by the collaborating laboratories. Experiments to
determine FRT-recombination rates in the mutant alleles, and gene expression
studies are also discussed.
159
ABSTRACT 121
THE
SANGER
MOUSE
GENETICS
THROUGHPUT CHARACTERISATION
PHENOTYPES IN KNOCKOUT MICE
Mark Lucas, C Podrini, E Cambridge, S Clare, L
of the Mouse Genetics Programme
The Sanger M.G.P., Wellcome Trust Sanger
PROGRAMME: HIGH
OF IMMUNOLOGICAL
Mottram, JK White on behalf
Institute, Genome Campus,
The Sanger Institute Mouse Genetics Programme is committed to making a
significant contribution to the functional annotation of the mammalian genome.
We are using the growing knockout first conditional ready targeted ES cell
resource currently being created at Sanger, to generate, characterise and archive
200 lines of knockout mice per year. In addition to studying the role of each
gene in normal development and function, the breadth and depth of our
phenotyping platform ensures that phenotypic data on a spectrum of disease
conditions are obtained for each mouse line without the need for any prior
assumptions about function. One area of focus is the immune system. Whole
blood is collected from mice that have undergone a standardised battery of
phenotyping tests. Multi-staining FACS analysis is used to profile the major
immune cell populations from these immunologically naïve bloods, whilst
haematological abnormalities are assessed through complete blood counts.
Furthermore, the immunoglobulin profile of plasma from these mice is assessed.
A panel of 42 tissues and organs including the spleen, thymus and mesenteric
lymph nodes are collected at necropsy and processed through to paraffin blocks
for storage in our Biobank as a stable, long term resource. An additional cohort
of mice from each mutant strain is also subjected to pathogenic challenge to
assess susceptibility to two common pathogens, Citrobacter rodentium and
Salmonella enterica serovar Typhimurium. Complementing this analysis, we
identify the expression profile of each gene using the lacZ reporter gene,
expressed under the control of the endogenous promoter of the target gene. To
date the PBL FACS analysis has completed 197 mutant lines, with 14
demonstrating a phenotype (7.7% hit rate) and the Plasma Immunoglobulin
analysis has completed 44 mutant lines with 5 lines showing a phenotype
(12.8% hit rate). Here we present a summary of the phenotyping data available
to date including examples of novel findings from a subset of immunologically
altered mutant lines.
160
ABSTRACT 122
THE MOUSE REFERENCE GENOME ASSEMBLY: UPDATES AND
RESOURCES
Valerie A Schneider and DM Church on behalf of the Genome Reference
Consortium and NCBI Genome Annotation Team
NCBI, Bethesda, MD; The Wellcome Trust Sanger Institute, Hinxton,
Cambridge, UK
The Genome Center At Washington University, St. Louis, MO; EBI, Hinxton,
Cambridge, UK
The current version of the mouse reference genome, MGSCv37, is among the
highest quality genomes in existence. Released in 2007, this assembly
representing the C57BL/6J strain is comprised of >96% finished, clone-based
sequence with <1% of the genome consisting of gaps. This valuable resource
plays critical roles in murine research and enables comparative analyses with the
genomes of humans and other organisms. The reference genome also serves as
the framework for the assembly of other mouse genomes sequenced by various
short-read technologies. The many research advances facilitated by the reference
genome have led to the recognition that some regions of the mouse genome are
not optimally represented in the current assembly. Regions recalcitrant to
cloning in BAC vectors remain as gaps, while highly complex repetitive
structures have caused mis-assembly at others. Due to inter-strain variation,
some loci are insufficiently represented by the single strain of the reference
genome. Correcting genome problems and providing sequence representation
for these strain-variable regions is a major focus of the Genome Reference
Consortium (GRC), the group responsible for the mouse reference genome. The
GRC strives to make its work transparent and welcomes community input and
feedback. We will present examples of regions under review by the GRC and
discuss plans for future releases of the mouse reference genome. NCBI provides
bioinformatics support for the GRC and also performs annotation of the mouse
reference assembly through its genome annotation pipeline. Annotated features
include genes, RefSeq transcripts, repeats, genomic sequences not used in the
assembly and genomic, gene trap and MICER clones. NCBI also provides
annotation for several partial assemblies from other mouse strains in regions
with significant inter-strain variation. The NCBI MapViewer can be used to
view this annotation and permits the integrated display of maps and assemblies
that use different coordinate systems, while the CloneFinder tool can be used to
identify clones that map to a defined genomic region as specified by coordinate
or annotated feature. We will present information on these tools and on recent
NCBI efforts to re-annotate MGSCv37.
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ABSTRACT 123
DBVAR AND DBSNP: NCBI DATABASES OF SIMPLE AND
STRUCTURAL VARIATIONS
Valerie Schneider, Tim Hefferon, John Garner, Azat Mardanov, Ming Ward,
Aleksey Vinokurov, Melvin Quintos, Mike Kholodov, David Shao, John Lopez,
Steve Sherry, and Deanna Church
National Center for Biotechnology Information, National Library of Medicine,
Bethesda, Maryland, USA
The National Center for Biotechnology Information (NCBI) creates and
maintains a set of databases that archive, process, display and report information
related to germline and somatic variants. These databases, the Database of
Genomic Structural Variations (dbVar) and the Database of Single Nucleotide
Polymorphisms (dbSNP), are integrated with many resources at NCBI including
Gene, GeneTests, OMIM, PubMed, and Nucleotide. This presentation focuses
on dbVar and dbSNP, summarizing current function and highlighting recent
improvements. Key to both databases are the archival and processing functions.
Each submission is assigned a database identifier (nssv# and ss#) based either on
flanking invariant sequence or locations asserted on reference sequences. Data
in dbSNP are then processed to aggregate information from multiple submitters
(assign rs#) and to calculate locations on each version of a genome and on NCBI
Reference Sequences (RefSeqs). Because these stable, public accessions are
citable in publications, they facilitate aggregation of information across studies.
Researchers and genetic testers are encouraged to submit their variation data and
to cite their submissions in manuscripts and on the web. Once data are
accessioned, they are made available in diverse ways: Entrez searches, studyspecific reports, annotation on the genome, human gene-specific displays such
as Variation Viewer, and ftp transfer. Advanced search functions to allow users
to find variants with specific qualities are available using our limits and
advanced search functions.
Acknowledgments
Work at NCBI is supported by the NIH Intramural Research Program and the
National Library of Medicine.
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ABSTRACT 124
CURRENT PROGRSSS OF PHENOTYPIC ANALYSES IN JAPAN
MOUSE CLINIC
Tomohiro Suzuki, Hideki Kaneda, Kimio Kobayashi, Ikuo Miura, Tamio Furuse,
Ikuko Yamada, Osamu Minowa, Hideaki Toki, Hiromi Motegi, Maki Inoue,
Tetsuo Noda, Hiroshi Maduya, and Sigeharu Wakana
RIKEN BioResource Center, Japan
Japan Mouse clinic (JMC) has been established for comprehensive mouse
phenotyping with national mouse resources since 2008. As far as we set up
phenotyping pipeline, standard operating procedures (SOPs), and home-page to
receive a request as a first step, and high-through put mouse reproduction
system based on in vitro fertilization technique and high-through put genome
scanning system for checking and integration of mouse genetic background as a
second step, and data capture and publication system of mouse phenotypic
information as a last step. Until now we have received requests of
comprehensive phenotypic analysis about 112 strains and have performed about
47 strains. The phenotypic information about a part of the 47 strains, including
13 standard strains, has been published on a phenotypic viewing site named
Pheno-Pub on JMC homepage (http://phenopub.brc.riken.jp/). In this meeting
we will show the results obtained from enormous phenotypic data in JMC about
variances among trials on all test items in JMC using phenotypic data obtained
from C57BL/6J, phenotypic differences between C57BL/6 sub-strains and
among breeding colonies, and frequencies of phenotypic differences among
genotypes (homozygote, heterozygote, and wild-type). In addition, we will
introduce the future plan about the construction of phenotyping platform to
analyze gene-environmental interactions between gene-function and
environmental factor such as nutrition, facility condition, and various stress for
understanding genetic predispositions for environmental influences.
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ABSTRACT 125
HYPERACTIVE MICE SHOW A CLUSTER OF ASSOCIATED
METABOLIC FEATURES
Jacqueline K White, Anna-Karin Gerdin, Christine Podrini on behalf of the
Mouse Genetics Programme, and Karen P Steel
Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK
Mutant mice with balance problems due to vestibular system defects show a
characteristic behavioural response of head-bobbing, circling and apparent
hyperactivity. They have long been known to have smaller body weight,
presumably due to their increased rate of exercise. We have taken a selection of
these mutant lines through our primary phenotypic screen to ask what other
features might be affected. The lines used included mutations in Myo6, Myo7a,
Cdh23, Grxcr1 and Mir96, among others. The mutant lines with hyperactivity
showed a characteristic cluster of other features with high frequency. Most
showed hearing impairment, low body weight, reduced fat mass, reduced plasma
cholesterol and lipoprotein levels, improved tolerance of glucose in the
intraperitoneal glucose tolerance test, increased latency to respond to heat in the
hot plate test, and increased activity, VO2 and VCO2 in the indirect calorimetry
test. A separate group of mutants selected for deafness alone without a balance
problem showed none of these features. A further set of mutant lines selected
for small body weight showed only reduced fat mass and increased VO2 and
VCO2, and none of the other features shown by the hyperactive group. The
genes involved are expressed in a variety of tissues with the only common site
being the inner ear. These data support a causal relationship between
hyperactivity and the other metabolic features observed.
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ABSTRACT 126
MULTIPLICITY OF PHENOTYPES IN EARLY GENERATIONS OF
THE MOUSE DIVERSITY OUTCROSS POPULATION
Karen L Svenson and GA Churchill
The Jackson Laboratory, Bar Harbor, Maine USA
The Diversity Outcross is currently being developed at The Jackson Laboratory
as a new mouse resource population. The DO is a heterogeneous mouse stock
derived from partially inbred ORNL Collaborative Cross strains. The population
utilizes a randomized outcrossing strategy with avoidance of sibling matings.
The DO is designed to compliment the CC strain panel by providing a high
resolution mapping resource. Groups of DO will approximate the genetic
diversity found in human populations. Each individual in the DO will be
genetically unique, representing a novel combination of alleles present in the
eight CC founder strains. Although individual DO animals cannot be replicated,
mapping results can be validated and permanent models constructed using the
CC strains or their F1 progeny. The randomized outcrossing scheme used to
create and maintain the DO population confers significantly greater genetic
variation than that found in common outbred strains of mice. The JAX DO is
currently producing the fifth outcross generation and is being maintained as 160
breeding pairs. We have obtained animals from the third outcross population and
have collected over 50 phenotypic measurements under both standard chow and
high fat feeding conditions. Many of these measurements describe features of
the metabolic syndrome, namely plasma glucose, insulin, triglycerides and HDL
cholesterol, and body composition (% fat and weight). As expected, DO animals
display a wide spectrum of variation in these and other parameters, including
whole blood analysis and behavioral traits such as home cage nesting and
aggressiveness. The phenotypic variation seen in the DO is comparable to that
observed in a large-scale inbred mouse strain survey and mimics that of human
populations. With rapidly emerging strategies for high throughput sequencing of
both DNA and RNA, the assignment of coincident and relevant disease
phenotypes to causative functional variants will also be accelerated, thereby
enhancing the development of well defined disease models.
165
ABSTRACT 127
SDOP-DB: A DATABASE FOR INTERNATIONAL SHARING AND
STANDARDIZATION OF MOUSE PHENOTYPING PROTOCOLS
Nobuhiko Tanaka1, Kazunori Waki1, Hideki Kaneda2, Tomohiro Suzuki2, Ikuko
Yamada2, Tamio Furuse2, Kimio Kobayashi2, Hiromi Motegi3, Hideaki Toki3,
Maki Inoue3, Osamu Minowa3, Tetsuo Noda3, Shigeharu Wakana2, and Hiroshi
Masuya1
1
Technology and Development Unit for Knowledge Base of Mouse Phenotype,
BRC, RIKEN, Japan, 2Technology and Development Team for Mouse Phenotype
Analysis, JMC, BRC, RIKEN, Japan, 3Team for Advanced Development and
Evaluation of Human Disease Models, BRC, RIKEN, Japan
Because differences in data between different protocols have often serious
effects on their derived results, there are crucial needs for sharing detailed
contents of protocols to integrate experimental data in experimental research
communities. For that purpose, we developed a new data format termed
Standardized Description of Operating Procedures (SDOPs), which provides an
assay-specific descriptive framework and enables direct and detailed comparison
of procedural parameters. Using this data format, we further developed a
comparative standardized-protocol database, SDOP-DB (http://www.brc.
riken.jp/lab/bpmp/SDOP/), as a practical application to share, compare and
evaluate the contents of protocols to enable data comparisons and analyses in the
field of mouse phenotypic analyses (Bioinformatics, 26(8), 1133-1134, 2010).
The SDOP-DB has the following features: (1). The SDOP-DB is helpful for
identifying procedural parameter(s) that can result in differences in data between
different protocols. Users can easily access phenotype data through hyperlinks to
mouse phenotype databases such as ‘Pheno-Pub’ (http://www.brc.riken.jp/
lab/jmc/mouse_clinic/en/m-strain_en.html) in Japan Mouse Clinic and
‘EuroPhenome’ (http://www.europhenome.org/databrowser/baselineViewer.jsp
) in European Mouse Disease Clinic, allowing association of the data with each
user's own in-house data.(2). For all analyses to be covered in SDOP-DB, users
can submit in-house standardized protocols by filling in MS-Excel form
downloaded from the website. Therefore, SDOP-DB can act as a web-based
repository of mouse phenotypic protocols. (3). Users can freely download SDOP
system from the website, enabling users to use as the application tool for
protocol version management both within and between laboratories. The many
uses of the system in the research community can accelerate standardization of
mouse phenotyping protocols.(4). To provide an interoperable framework for
sharing and reuse of mouse phenotyping protocols among laboratories, we
provide the SDOP-DB data in the Phenotyping Procedures Markup Language
(PPML) format, a standardized data format for describing experimental
protocols developed by Mouse Phenotype Database Integration Consortium
(InterPhenome). The above-described features in the SDOP-DB are helpful for
the international sharing and standardization of mouse phenotyping protocols.
166
ABSTRACT 128
MODELING HUMAN CHROMOSOME 21-ASSOCIATED MENTAL
RETARDATION DISORDERS IN MICE
Tao Yu1, Zhongyou Li1, Steven J Clapcote2, Zhengping Jia2, Chunhong Liu1,
Annie Pao1, Sei-ichi Matsui1, Norma J Nowak1, John C Roder2, Chu Chen3,
Allan Bradley4, and Eugene Y Yu1
1
Roswell Park Cancer Institute, Buffalo, NY, USA
2
University of Toronto, Toronto, Canada
3
Louisiana State University, New Orleans, LA, USA
4
Wellcome Trust Sanger Institute, Cambridge, UK
Copy-number variation in the human genome can be disease-causing or
phenotypically neutral. This type of genetic rearrangement associated with
human chromosome 21 (Hsa21) includes partial Monosomy 21 and Trisomy 21,
which are genomic causes for intellectual disability. The regions on Hsa21 are
syntenically conserved with approximately 2.3, 22.9 and 1.1-Mb regions located
on mouse chromosome 10 (Mmu10), Mmu16 and Mmu17, respectively. In this
project, we generated chromosomal deletions and the reciprocal duplications
spanning the entire Hsa21 syntenic regions on Mmu10, Mmu16 and Mmu17 in
mouse ES cells using Cre/loxP-mediated chromosome engineering, which led to
viable mutant mice carrying the corresponding deletions on Mmu10 or Mmu17
as well as duplications on Mmu10, Mmu16, or Mmu17. The phenotypic analysis
of the mutant mice carrying individual rearrangement or compound
rearrangements shows intellectual disability-related phenotypes. Mutant mice
carrying either deletion exhibit impairment in learning and memory. The mutant
mice trisomic for all Hsa21 syntenic regions exhibit Down syndrome-related
neurological defects, including impaired cognitive behaviors, reduced
hippocampal long-term potentiation and hydrocephalus. These results suggest
that when all the mouse orthologs of the Hsa21 genes are triplicated, an
abnormal cognitively relevant phenotype is the final outcome of the elevated
expressions of these orthologs as well as all the possible functional interactions
among themselves and/or with other mouse genes. Because of the desirable
genotypes and phenotypes, these deletion and duplication mouse models may
serve as powerful tools for further understanding intellectual disability
associated with partial Monosomy 21 or Trisomy 21 and may also be used for
developing novel therapeutic interventions for the clinical manifestation of the
disorders.
167
ABSTRACT 129
TAIWAN MOUSE CLINIC—AN EXPERIMENT FOR
PHENOTYPING SERVICE
Jeffrey Jong-Young Yen, Yen-Hui Chen, and Ya-Wen Hsaio
Taiwan Mouse Clinic, NRPGM, and IBMS, AS, Taipei, Taiwan
MOUSE
The mouse has emerged as one of the most popular human disease models
recently since the availability of enormous amount of mutant mouse lines
generated by spontaneous mutation, induced mutagenesis, transgenics and
insertional mutagenesis, and gene targeting strategy. However, it is also well
recognized that capitalizing of these mutant resources critically dependents upon
our capability of comprehensive phenotyping for general health and in-depth
characterization for a special physiological function. Since May 2008, we have
established a comprehensive mouse phenotyping core facility in Taipei, called
Taiwan Mouse Clinic, to promote local biomedical researches using the mouse
as model by the funding through the National Research Program on Genomic
Medicine (NRPGM), NSC, Taiwan. There are three major goals of our project:
one, to construct a centralized, modular and user-friendly mouse phenotyping
platform; two, to provide services, education and consultation for both entrylevel general physical checkup and advance phenotyping techniques; three, to
provide a unique local, national and international resource for studying mouse
models of human diseases. The key component of this project is the
establishment of the Mouse Hotel which honors the health certificates from all
mouse vivaria and only requests a minimum essential specific pathogen list and
greatly reduces the turn-around time of animal trafficking within mouse clinic.
All animals in hotel are all-in and all-out and are handled as if in P2 vivarium,
and both animals and equipments in phenotyping laboratories are monitored for
pathogen contamination routinely. In the past one and half year there are more
than 50 laboratories all over Taiwan requested our services and the regular
services have grown up to 5000 items per month and have reached more than
78% of our maximum service capacity. We have also obtained the base line
comprehensive phenotyping data for the C57BL/6JNarl strain and have revised
the standard operational procedures for home cage activity analysis with Clever
System and for metabolic rate analysis with LabMaster Calorimetry Module.
Finally, we have engaged enthusiastically in regional and international
collaboration in exchanging phenotyping platforms, databases, and resources
through the networks of Asia Mouse Phenotyping Consortium (AMPC) and
International Mouse Phenotyping Consortium (IMPC).
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ABSTRACT 130
INITIAL ASSEMBLY OF 17 MOUSE STRAINS FROM THE MOUSE
GENOMES PROJECT
Guy Slater1, Thomas M Keane1, Jared Simpson1, Aylwyn Scally1, Xiangchao
Gan2, Richard Mott2, Jonathan Flint2, and David J Adams2
1
Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK
2
Wellcome Trust Centre for Human Genetics, Oxford University, Oxford, UK
The Mouse Genomes Project has sequenced the genomes of 17 inbred mouse
strains to between 20-35x coverage using paired-end Illumina sequencing. The
reads vary in length from 36 to 108bp, and the read pairs contain insert sizes
ranging from 150bp to 3kb. We present initial sequence assemblies of these 17
mouse strains. Assembly of such datasets remains challenging and a pure
denovo approach is impractical. Therefore, we have developed a low memory
assembly pipeline using a reference guided approach, whereby read pairs are
mapped to the reference and then split into groups corresponding to the region to
which they have been mapped. These groups of reads were de novo assembled
using velvet, and the resulting contigs are refined by filling gaps between
contigs by using alignment-guided local reassembly. For each strain, there is a
varying sized pool of reads that do not map to the C57BL/6J reference and these
are assembled de novo separately. The initial results from the assembly pipeline
produced contigs with an N50 of approx.5kb. We are then using a mixed
scaffolding approach that uses a combination of the reference and longer insert
reads (3kb-40kb) to produce chromosome sized scaffolds per strain. The quality
of the assemblies are evaluated by comparison of the C57BL/6NJ assembly to
the C57BL/6N reference, and also by comparison of the NOD assembly to
10Mbp of manually finished BACs. We estimate that between 2-18Mbp of
sequence is novel and not contained in the reference sequence, with some novel
contigs being up to 10kbp in length and containing novel protein coding
sequences.
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ABSTRACT 131
CLUSTERED PROTOCADHERIN GENES: GENETIC CODES FOR
GENERATING THE COMPLEX BRAIN
Takeshi Yagi, Shunsuke Toyoda, Yasushi Itoga, Keizo Hirano, Atsushi Okayama,
Shinichi Yokota, Shota Katori, Teruyoshi Hirayama, and Takahiro Hirabayashi
Osaka University, Graduate School for Frontier Biosciences, Lab of KOKOROBiology, Japan
The brain is a complex system composed of enormous numbers of differentiated
neurons. To examine the molecular mechanisms underlying complex
organization of the brain, it is important to identify the molecules involved in
generating neural diversity and organization. The clustered protocadherin (Pcdh)
genes encode diverse cadherin-related proteins that express in the brain. In
mammals, the clustered Pcdh family consists of three gene clusters: Pcdh-a,
Pcdh-b, and Pcdh-g. At the single-cell level, several dozen Pcdh-a, -b and -g
mRNAs are regulated monoallelically, resulting in the combinatorial expression
of distinct variable exons. The PCDH-Aand PCDH-G proteins also form
oligomers, further increasing the molecular diversity at the cell surface. Loss-of
function mice in the clustered Pcdh genes had neurological abnormalities for
generating neural networks. Also we found epigenetic regulations of clustered
Pcdh genes in the individual neurons. Here I would like to discuss genetic codes
for generating the complex brain system.
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ABSTRACT 132
COMPREHENSIVE PHENOTYPING OF MOUSE MODELS
Tania Sorg, Marie-France Champy, Elodie Bedu, Roy Combe, Hamid Meziane, and
Yann Herault
Institut Clinique de la Souris, Illkirch, France
The Institut Clinique de la Souris (ICS) / Mouse Clinical Institute (MCI) is a
technology platform that provides a comprehensive set of highly specialized mouse
services to scientists from academia and industry. The ICS combines the capacity of
generating mutant mice on a large scale with a high-throughput and comprehensive
phenotypic analysis of mice. The ICS phenotyping platforms are adapted for the
study of genetically engineered mouse models (GEMMs), but can also be used for
pharmacological and toxicological studies in the mouse.
The ICS services will ultimately help the scientific community to use the mouse to
develop a complete functional annotation of the human genome and to employ this
to better understand human diseases and their underlying physiological and
pathological basis. Achieving this goal will underpin and speed the
biopharmaceutical and biotech industries that increasingly depend upon advanced,
mouse-based analysis in drug development.
The ICS has successfully assembled a comprehensive phenotyping platform, which
at present is composed of the 5 following core units:
- Clinical Chemistry laboratory. The activities of this laboratory cover clinical
chemistry, hematology, coagulation, immunology, endocrinology and other
metabolites.
- Metabolic exploration. This service is set up to phenotype the metabolic function,
such as in diabetes and obesity models. Currently tests are in place to analyze the
body composition, glucose homeostasis, and energy expenditure, as well as the
skeleto-muscular and the uro-genital systems, and the gastro-intestinal tract.
- Cardiovascular and respiratory exploration. This service is set up to phenotype
the function of the cardio-respiratory system. Currently tests are in place to analyze
the cardiac function and anatomy, as well as the respiratory system such as in asthma
models.
- Behavior and nervous system. This service explores the central and peripheral
nervous system, sensory systems, as well as behavior in mice. This core has
developed a comprehensive tests battery to evaluate: general CNS function, affective
behaviors (anxiety, depression), cognitive function, sensory thresholds and
analgesia. Assays have also been set up to analyze the sensory systems (visual and
auditory functions)
- Histology and pathology. This service provides a comprehensive histological and
histopathological analysis of mutant and control mice, as well as embryology
studies.
The assays performed by each core unit will be presented, as well as validated flow
schemes for applications in therapeutic areas.
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ABSTRACT 133
TARGETED AND WHOLE EXOME RESEQUENCING OF MOUSE
MONOGENIC MUTANTS, QUANTITATIVE TRAIT LOCI AND
CANCER MODELS
David R. Beier1, Jennifer L. Moran2, Evan Mauceli2, Snaevar Sigurdsson2, Tim
Fennell2, Lauren Ambrogio2, Miriam H. Meisler3, John C. Schimenti4, Karen
Cichowski1, Laura Reinholdt5, Jane Wilkinson2, Stacey B. Gabriel2, Federica di
Palma2, and Kerstin Lindblad-Toh3
1
Genetics Division, Brigham & Women’s Hospital, Harvard Medical School,
Boston, MA, USA
2
The Broad Institute, Cambridge, MA, USA
3
Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA
4
Center for Vertebrate Genomics, Cornell University, Ithaca, NY, USA
5
We have established a mouse mutant resequencing initiative in order to apply
next generation sequencing technology to the identification of ENU-induced and
spontaneous mutations, quantitative trait loci (QTL) and cancer mutations.
Funding of the initiative at the Broad Institute through The Large-Scale Genome
Sequencing Program [National Human Genome Research Institute (NHGRI);
NIH] has enabled pilot studies to be performed and future sequencing of
approved models to be free to the mouse community. In two pilot studies,
targeted resequencing of 13 monogenic mutants mapped to <5 Mb intervals was
performed by solution-based hybrid selection of genomic DNA libraries and
Agilent oligonucleotide RNA baits followed by Illumina sequencing. For
spontaneous mutants and ENU-induced mutants in which a mutation in a noncoding region was suspected, the entire non-recombinant intervals were
sequenced in mutant and control strains. For most ENU-induced mutants, all
RefSeq exons, UTRs and promoters in non-recombinant intervals were
sequenced. Over 5 Mb of genomic DNA covering 565 genes was sequenced in
pilot work. Of 9 ENU-induced mutants analyzed, causal mutations were
identified in 5 mutants. We have now designed two mouse exome baits covering
all coding regions of a set of ~21,500 curated genes on the mm9 genome
assembly. The more optimal exome design will be used in production for this
initiative at the Broad and will also be available to the community through
purchase from Agilent. An application process will be implemented for 1)
sequencing of cancer models and mapped or unmapped monogenic mutants by
whole exome sequencing and 2) Nimblegen-based targeted genomic regionbased sequencing of mutants or mapped quantitative trait loci.
172
ABSTRACT 134
DYNAMIC
TRANSCRIPTOMES
DURING
NEURAL
DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS
REVEALED BY SHORT, LONG, AND PAIRED-END SEQUENCING
Jia Qian Wu 1, Lukas Habegger2, Parinya Noisa3, Anna Szekely4, Caihong Qiu5,
Stephen Hutchison6, Debasish Raha7, Michael Egholm6, Haifan Lin5, Sherman
Weissman4, Wei Cui3, Mark Gerstein2,8,9, and Michael Snyder1
1
Department of Genetics, Stanford University School of Medicine, Stanford, CA
2
Program in Computational Biology and Bioinformatics, Yale University, New
Haven, CT
3
Institute of Reproductive and Developmental Biology, Imperial College
London, London, UK
4
Genetics Department, Yale University, New Haven, CT
5
Yale Stem Cell Center, Yale University, New Haven, CT
6
454 Life Sciences Sequencing Centre, Branford, CT
7
Molecular, Cellular and Developmental Biology Department, Yale University,
New Haven, CT
8
olecular Biophysics and Biochemistry Department, Yale University, New
Haven, CT
9
Computer Science Department, Yale University, New Haven, CT
In order to examine the fundamental mechanisms governing neural
differentiation, we analyzed the transcriptome changes that occur during the
differentiation of human embryonic stem cells (hESCs) into the neural lineage.
Undifferentiated hESCs as well as cells at three stages of early neural
differentiation, N1 (early initiation), N2 (neural progenitor), and N3 (early gliallike) were analyzed using a combination of single read, paired-end read, and
long read RNA sequencing. The results revealed enormous complexity in gene
transcription and splicing dynamics during neural cell differentiation. We found
previously unannotated transcripts and spliced isoforms specific for each stage
of differentiation. Interestingly, splicing isoform diversity is highest in
undifferentiated hESCs and decreases upon differentiation, a phenomenon we
call “isoform specialization.” During neural differentiation, we observed
differential expression of many types of genes including those involved in key
signaling pathways, and a large number of extracellular receptors exhibit stagespecific regulation. These results provide a valuable resource for studying neural
differentiation and reveal new insights into the mechanisms underlying in vitro
neural differentiation of hESCs, such as neural fate specification, NPC identity
maintenance and the transition from a predominantly neuronal state into one
with increased gliogenic potential.
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ABSTRACT 135
IMPROVING MOUSE GENOME ANNOTATION THROUGH NEW
TECHNOLOGIES
Laurens Wilming, Gary Saunders, Markus Brosch, Gavin Laird, Adam
Frankish, and Jennifer Harrow
Wellcome Trust Sanger Institute, Cambridgeshire, UK
New technologies - RNA-seq, protein mass spectrometry and multiple genome
sequencing - provide researchers with a treasure trove of new data. Examining
gene families such as MUP (Major Urinary Protein) or regions of interest such
as the MHC (Major Histocompatibility Complex), we compare how the Mouse
Genomes project (sequencing 17 mouse inbred strains) performs in these
complex regions. New RNA-seq data is helping us identify new transcribed
regions and offers the ability of correlating splice variants with tissues, cell
types or developmental stages. Mouse proteome data produced by WTSI,
combined with Peptide Atlas data, revealed over 10 new protein-coding genes
not present in any database, and also evidence for exapted pseudogene loci that
produce peptides. New proteome and transcriptome data allows us to complete
and refine annotation by indicating new variants and extensions to incomplete
genes and by showing previously separate gene fragments that can be joined into
single loci. Apart from aiding annotation in general, this also helps us resolve
CCDS (Consensus Coding Sequence project) conflicts.
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ABSTRACT 136
GENOMIC ALTERATIONS IN TUMORS OF THE MCM4CHAOS3
BREAST CANCER MOUSE MODEL
Marsha D. Wallace1, Ethan Cerami2, Stefan Stefanov1, Lishuang Shen1, and
John C. Schimenti1
1
Cornell University, Ithaca, NY, USA
2
Memorial Sloan-Kettering Cancer Center, New York, NY, USA
Our Chaos3 model in the defined C3H background contains the only
endogenous gene mutation in mice known to lead exclusively to mammary
tumors. Chaos3 is a point mutation of a highly conserved residue in the
minichromosome maintenance 4 (Mcm4) gene, detected in a screen for Genomic
Instability (GIN). MCM4 is a subunit of the Mcm2-7 complex that acts as the
helicase to unwind DNA during DNA replication. It is also required for DNA
replication licensing, which restricts replication to only once per cell cycle.
When homozygous Chaos3 female C3H mice are aged, >80% develop
exclusively breast cancer at ~12 months. To determine the underlying
biological basis by which a point mutation in a DNA replication gene ultimately
leads to breast cancer, we have employed Nimblegen Sequence Capture
technology and CGH arrays to analyze Chaos3 tumors. We designed a Seq Cap
chip containing ~1200 candidate cancer related genes to find mutations
contributing to carcinogenesis. Furthermore, we analyzed Copy Number
Variations (CNVs) in 11 Chaos3 tumors using Nimblegen 720K CGH arrays.
While few mutations were uncovered, interestingly we find CNVs shared among
many Chaos3 tumors. Virtually all Chaos3 tumors shared amplified regions on
Chromosome 12 and 16. Also, specific regions on Chromosome 4, 5, and 11 are
deleted in the majority of Chaos3 mammary tumors specifically, suggesting
deletion of genes in these regions may be responsible for driving mammary
transformation in the model. These genes are excellent candidates for future
studies to validate susceptibility genes underlying spontaneous or heritable
forms of breast cancer, demonstrating Chaos3 mice are a unique and powerful
model to investigate mammary tumor carcinogenesis.
175
ABSTRACT 137
A MISSENSE MUTATION IN THE NOVEL WD-REPEATCONTAINING PROTEIN, WDR81, CAUSES PURKINJE CELL AND
PHOTORECEPTOR CELL DEATH IN THE ENU-INDUCED
NEUROLOGICAL MOUSE MUTANT NUR5
Maria Traka1, Kathleen J. Millen2, and Brian Popko1
1
Department of Neurology, The University of Chicago Center for Peripheral
Neuropathy, The University of Chicago, Chicago, USA
2
Department of Human Genetics, The University of Chicago, Chicago, IL, USA
To identify genes that affect neuronal survival in the central nervous system
(CNS), we characterized the N-ethyl-N-nitrosourea-(ENU) induced autosomal
recessive mutant mouse line nur5, which develops an adult-onset motor
coordination disorder that significantly worsens with age. Histological analysis
of these mice showed progressive postnatal Purkinje cell and photoreceptor cell
death.
The nur5 mutation was initially localized to a 24 cM region of
Chromosome 11 between the Trp53 and Wnt3 genes. To fine-map the mutation,
we performed genetic linkage analysis of polymorphic markers at the Trp53 Wnt3 interval that identified an L302P mutation in the WD-repeat-containing
protein, WDR81. We confirmed that the nur5 phenotype is caused by the
mutation in the Wdr81 gene by transgenic BAC rescue. Therefore, Wdr81nur5
was designated as a mutant allele of the Wdr81 gene. We show that WDR81 is
expressed in Purkinje cells and photoreceptor cells, as well as other CNS
neurons, and its expression pattern, based on immunohistochemical analysis, is
not affected by the Wdr81nur5mutation. EM analysis demonstrated a prominent
accumulation of large membranous particles in the mutant Purkinje cell
dendrites that precedes neuronal loss, suggesting that dendrite defects may result
in Purkinje cell death in Wdr81nur5 mutants. To discover potential binding
partners of WDR81, we performed a yeast-two hybrid screen and coimmunoprecipitation analysis that revealed an association of this protein with
the spectrin-repeat containing protein SYNE1. SYNE1 is expressed in Purkinje
cells and is known to be important for Purkinje cell physiology, as the premature
termination of SYNE1 was recently identified to be the cause of human adultonset autosomal recessive ataxia type 1 (ARCA-1) with cerebellar atrophy. The
association of WDR81 with SYNE1 suggests that these proteins are part of a
multimolecular complex that is critical for neuronal survival.
176
ABSTRACT 138
USING WHOLE GENOME NEXT-GENERATION SEQUENCING AS A
TOOL TO FIND ENU-INDUCED MUTATIONS
Michelle Simon, S Greenaway, P Denny, A-M Mallon, and JM Hancock
Medical Research Council, Mammalian Genetics Unit, Harwell, Oxfordshire,
U.K.
Phenotype-driven screens after chemical mutagenesis of males with N-ethyl-Nnitrosourea (ENU) at MRC Harwell have been incredibly productive in
identifying mouse mutants of biological interest. Nevertheless, identification of
the causative mutations by conventional linkage analysis and Sanger sequencing
of genes in the minimal genetic interval remains a bottleneck. We have been
using next generation sequencing, using the Illumina Genome Analyser 2x
platform, to accelerate this process of mutation detection. A sequence analysis
pipeline, based on existing packages (i.e. Bowtie and Samtools) has been used
to align reads to the C57BL/6J reference sequence, automatically identify
unique variants, populate a custom sequence database and identify low and high
confidence SNPs. Further developments will include assessment of the impact
of putative mutations on splicing efficiency, predicted protein structure and
integration with Mousebook – the MRC Harwell web portal. We will present the
design and implementation of the re-sequencing pipeline, the methodology used
to categorise high confidence SNPs and the identification of novel natural
variants.
177
ABSTRACT 139
THE
SANGER
MOUSE
GENETICS
PROGRAMME; HIGH
THROUGHPUT MORPHOLOGICAL ANALYSIS OF THE EYE
Valerie E Vancollie1, J Estabel1, D Sunter1, VB Mahajan2,3, SH Tsang4, MC
Naumann4, M Mahajan2,3, JK White1, on behalf of the Mouse Genetics
Programme
1
The Sanger Mouse Genetics Programme, Wellcome Trust Sanger Institute,
2
Department of Ophthalmology and Visual Sciences, University of Iowa, IA USA
3
Omics Laboratory, University of Iowa, IA USA
4
Bernard and Shirlee Brown Glaucoma Laboratory, Department of Pathology
and Cell Biology AND Department of Ophthalmology, College of Physicians
and Surgeons, Columbia University, New York, NY USA
The Sanger Mouse Genetics Programme aims to make a significant impact on
our understanding of the function of genes and their role in disease by
generating, characterising and archiving in the order of 200 lines of knockout
mice per year. Knockout mice are phenotyped using a battery of tests relevant to
key disease areas including diabetes, obesity, hearing and vision disorders,
immune disorders, pain and motor function. In addition to studying the role of
each gene in normal development and function, the breadth and depth of our
phenotyping platform ensures that phenotypic data on a spectrum of disease
conditions are obtained for each mouse line without the need for any prior
assumptions about gene function. Here we will report on the utility of our high
throughput approach for the morphological examination of eyes. Eyes are first
screened using a slit lamp following a standardised list of parameters in order to
view the lids, cornea, pupil and iris. Eyes are then dilated to view the lens and
images of abnormalities are taken with a slit lamp mounted camera. The screen
continues with eyes in the dilated state using an ophthalmoscope to view the
retina. Any abnormalities observed are then imaged using Topical Endoscopic
Fundus Imaging. Complementing this analysis, we identify the expression
profile of each gene in adults and at E14.5, using the lacZ reporter gene,
expressed under the control of the endogenous promoter of the target gene.
Through collaboration, detailed histological analysis of all knockout lines that
enter our pipeline is performed with follow up analysis on lines where lacZ
expression has been noted in eye wholemounts. As a result of the combination
of our core high-throughput eye morphology screen and collaboration on
histological analyses we have observed phenotypic abnormalities in ~7% of all
mutant lines examined to date.
178
ABSTRACT 140
A
CELLULAR
GENETICS
PLATFORM
TO
IDENTIFY
PHARMACOGENETIC TOXICITY PATHWAYS
O Suzuki1, N Butz1, M Pletcher2, A Su3, B Steffy1, D Scoville1, A Frick1, J Trask4,
R Thomas4, and Tim Wiltshire1
1
University of North Carolina at Chapel Hill, Division of Pharmacotherapy and
Experimental Therapeutics
2
Pfizer Incorporated Groton, Connecticut
3
Genomics Institute of Novartis Research Foundation at La Jolla, California
4
The Hamner Institute for Health Sciences, Research Triangle Park, North
Carolina, USA
Pharmacogenetic studies have successfully identified genetic variants that
contribute to variation in susceptibility to drug responses, but it is still a complex
and challenging task to evaluate broadly across the human genome to identify
the genetic components of response to drugs. Despite limitations there is a
pressing need to identify genetic components that contribute to the efficacy and
toxicity of drugs, across a wide spectrum of agents. We have proposed that
using an alternative model population approach will provide many underlying
mechanisms and pathways that are implicated in drug activity and responses.
We have developed a platform of both in-vitro and in-vivo assays from
genetically well defined mouse strains which will enable us to assess effects of
toxicity and efficacy of current and novel agents in drug therapies. Here, we
present results obtained using a new strategy of cellular genetics to identify
genes and gene pathways that underlie susceptibility to cellular-level adverse
drug reaction and toxicities. We have the ability to pinpoint genetic components
that contribute to, or underlie, the toxicity phenotypes. We have screened 75
toxicants/drugs in a high-content imaging screen that determines changes in
specific cell-health status phenotypes (nuclear changes, membrane permeability,
mitochondrial membrane potential and apoptosis). Primary cell lines from 32
inbred mouse strains were phenotyped and the drug responses were used in
genome-wide association analyses to identify loci that underlie the toxicity
traits. We identify a number of potential candidate genes for specific drug
toxicity phenotypes and provide validation of targets using over-expression and
siRNA knockdown of specific gene targets. Genes were selected and prioritized
for validation based on network analysis and expression in mouse embryonic
fibroblasts. In addition, we identify broader cross-drug toxicity pathways.
Gene-set enrichment analysis of the candidate loci revealed an overrepresentation of genes involved in cell-cycle regulation.
179
ABSTRACT 141
LESSONS FROM PHENOTYPE OF KO MOUSE, THE ROLE OF
SNUR4 ON ADIPOGENESIS AND INSULIN SENSITIVITY
Jae Hoon Shin1, Il Yong Kim1, Yo Na Kim1,2, Ji Won Choi1, Kyung Jin Roh1, Mi
Ra Sohn4, Cheol soo Choi2,3, Yun Soo Bae4, and Je Kyung Seong1
1
Laboratory of Developmental Biology and Genomics, College of Veterinary
Medicine, Seoul National University, Seoul, Korea
2
Lee Gil Ya Cancer and Diabetes Institute, 3Division of Endocrinology Gil
Medical Center, Gachon University of Medicine and Science, Incheon
4
Laboratory of Molecular Biochemistry Division of Molecular Life Sciences
Ewha Woman’s University, Seoul, Korea
Recently we developed SNUR4 KO mice. The body weight of SNUR4 KO
mice were decreased with growth retardation due to decreased abdominal body
fat. To validate the anti-obesity role of SNUR4, HFD (High fat diet) and LFD
(Low fat diet) were fed for 12 weeks to SNUR4 knock-out (KO) mice (-/-),
hetero KO (+/-), and wild-type mice. The body weight of HFD-fed SNUR4 KO
mice showed significantly reduced with the rate of weight gain compared to
HFD-fed wild-type mice. The ratio of epididymal fat weight to body weight and
adipocyte size significantly decreased in HFD-fed SNUR4 KO compared with
the HFD-fed wild-type mice. However, the fatty liver change was observed both
two groups. Also the expression level of transcriptional factors and enzymes
leading to lipogenesis including Acrp30, aP2, PPARr, FAS, and SREBP was
decreased in adipose tissue and liver from HFD-fed SNUR4 KO mice. Next, we
test metabolic phenotyping using mouse metabolic phenotype system to identify
the cause of resistance to diet-induced obesity in SNUR4 KO mice. To
determine metabolic rate in SNUR4 KO, we measured oxygen consumption
(VO2), respiratory exchange ratio (RER) and energy expenditure. There are no
differences between SNUR4 KO and wild-type mice in RER and energy
expenditure. Locomotor activity and food intake were also similar between two
groups. We performed hyperinsulinemic-euglycemic clamps to determine
insulin dependent glucose uptake in SNUR4 KO mice. The glucose infusion rate
was increased in SNUR4 KO mice than wild-type mice. Hepatic glucose output
was decreased in SNUR4 KO mice than wild-type mice in clamped condition
but no difference was detected in basal condition. SNUR4 KO mice were found
to take up significantly more glucose and this glucose into glycolysis and
glycogen synthesis. Our finding suggests that SNUR4 KO mice are resistance to
diet-induced obesity in adipose tissue. Also, insulin-stimulated glucose uptake
and metabolism were more sensitive in SNUR4 KO mice than wild-type mice.
180
ABSTRACT 142
EPIBLAST-ABLATION OF SOX2 EXPRESSION LEADS TO
NEUROCRISTOPATHIES IN MOUSE EMBRYOS
Nikolaos Mandalos1,*, Marannia Saridaki1*, Jessica Lea Harper1, Anastasia
Kotsoni1, Aris N. Economides2, and Eumorphia Remboutsika1
1
Stem Cell Biology Laboratory, Institute of Molecular Biology and Genetics,
Biomedical Sciences Research Centre “Alexander Fleming”, Vari, Greece
2
Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA
*equal contribution
Sox2 is a single exon gene essential for the maintenance of pluripotency, the
development of the epiblast and trachea, lung and nervous system development
and homeostasis. Precise genetic manipulations in the Sox2 locus to alter the
expression of Sox2 are essential due (a) to the fact that the proximal promoter
and the coding region of the Sox2 gene are embedded in a CpG island and (b) to
the presence of a miRNA (mmu-mir1897) and another non-coding RNA
transcript (Sox2ot) in the region. Here, we describe the ablation of Sox2 in the
epiblast using a novel Conditional by Inversion Sox2 allele (Sox2COIN).
Sox2COIN/COIN mice are normal and fertile and epiblast-specific inversion of the
Sox2COIN allele using Sox2-Cre generates normal heterozygote animals
(Sox2INV/+:TgSox2CRE). Sox2INV/+ intercrosses generate a true Sox2 null allele as
homozygote mice (Sox2INV/INV) are not viable and embryos die around
implantation. Sox2INV/mosaic embryos die around E11 and exhibit a range
craniofacial and heart defects as well as abnormal growth structures in the
central nervous system. These defects are described in human conditions known
as neurocristopathies and have been attributed to defects in neural crest
development. Our results demonstrate that COIN is a method of choice for
precise manipulations of single exon genes and reveals a novel role for Sox2 in
neurocristopathies.
181
ABSTRACT 143
A COMPREHENSIVE CATALOGUE OF STRUCTURAL VARIATION
FROM 17 INBRED MOUSE STRAINS
Kim Wong1,$ $B Yalcin2,$ $T Keane1,$ $M Goodson2,$ $A Agam2,$ $C Nellaker2,$ $J Stalker1,$
$J Flint2,$ $and D Adams1
1
The Wellcome Trust Sanger Institute,$ $Hinxton,$ $Cambridgeshire,$ $UK
1
Wellcome Trust Centre for Human Genetics,$ $Oxford,$ $UK
The genomes of 17 key inbred mouse strains have been sequenced by the
Mouse Genomes Project (http://www.sanger.ac.uk/mousegenomes/ ) using
next-generation sequencing technology. The strains, which include 4 ‬wildderived strains (CAST/EiJ, Spretus/EiJ, PWK/PhJ and WSB/EiJ ) and 13
laboratory strains (129P2/O1a, 129S1/SvImJ, 129S5, A/J, AKR/J, BALB/cJ,
C3H/HeJ, C57BL/6NJ, CBA/J, DBA/2J, LP/J, OD/ShiLtJ, NZO/HiLtJ ),
were sequenced to 20-35x depth. This has enabled us to compare all 17 strains
to the reference genome to identify a highly confident set of large (>100bp)
structural variants (SVs ) such as insertions, deletions, inversions, and copy
number gains. Currently, several methods are available to identify SVs from
mapped read data, however, independently, none are able to identify all types
and sizes of SVs. We have developed a pipeline, SVMerge, which enabled us
to utilize a suite of software tools to generate a more comprehensive set of SVs.
We have computationally validated each SV by performing local assembly,
which also enabled better localization of the breakpoints. We have performed
experimental validation on each class of automated calls to evaluate the false
discovery rates, and performed manual SV annotation to determine false
negative rates. In total, we have identified a non-redundant catalogue of over
400,000 SVs among the 17 strains. The majority of SVs in the laboratory
strains are shared with at least one other strain. The wild-derived strains,
however, harbor a greater number of unique SVs and more genes are disrupted
by SVs. Deletions, for example, disrupt approximately 200 protein-coding
genes in each laboratory strain, and up to 700 genes in the wild-derived strains.
Finally, by observing the strain distribution patterns of these SV events and
correlating them with inter-strain gene expression differences, we have been
able to determine the potential phenotypic consequences of individual SVs.
This is the most comprehensive collection of large, fine-scale mouse SVs to
date, and together with the complete assemblies, SNPs, and short indels, will
be a valuable resource for the mouse genetics community.
182
ABSTRACT 144
COLLECTION AND DISTRIBUTION OF CRE/FLP-DRIVERS AT THE
RIKEN BIORESOURCE CENTER
Atsushi Yoshiki, Kazuyuki Mekada, Hatsumi Nakata, Ayumi Murakami, Masayo
Kadota, Fumio Ike, Noriko Hiraiwa, Kaoru Fukami-Kobayashi, Yuichi Obata
RIKEN BioResource Center, Tsukuba, Japan
The RIKEN Bioresource Center (RBRC) is the core facility for mouse resources
in Japan under the National BioResource Project (NBRP, http://www.nbrp.jp) of
the Japanese Ministry of Education, Culture, Sports, Science, and Technology,
and is also a member of the International Coordination Group of the CREATE
consortium (http://www.creline.org). RBRC collects, develops, and distributes
useful Cre/Flp-drivers for functional genomics studies in collaboration with the
international scientific community. Currently, the characterization of the tissue
specific expression of these drivers and the associated phenotype data is a
challenging issue that will be used to enrich the value of the Cre/Flp-drivers.
RBRC has so far collected 73 Cre-drivers and 4 Flp-drivers, and has distributed
these lines worldwide via our website (http://www.brc.riken.jp/lab/animal/en),
the International Mouse Strain Resource (IMSR, http://www.findmice.org), and
Biomart (http://www.creline.org/search_cre_mice). The 73 Cre-drivers consist
of 59 transgenic and 14 targeted knock-in mice. The most popular lines include
deleter mice that are used in crosses with conditional knockout mice such as B6Tg(CAG-Cre)CZ-MO2Osb and B6-Tg(CAG-FLPe), and other tissue specific
Cre-drivers such as Emx1-Cre, Mesp1-Cre, Nestin-Cre, and Neurog3-Cre. In
addition, we are developing new Cre-drivers with different promoters, such as
Ins1, Krt14, Tagln, Tek/Tie2, Vil1, and Wap, as well as fluorescent reporters
using transgenesis and knock-in methods on the C57BL/6NCrlj background. In
this study, we report the updated list of these Cre/Flp-drivers with data for their
tissue-specificity and the status of our development program for Cre-drivers and
reporters at RBRC.
183
ABSTRACT 145
ANALYSIS OF THE EXPRESSION PROFILE OF CRH-POMC SYSTEM
GENES IN VITILIGO SKIN BIOPSIES
Ene Reimann1,2,, K Kingo2, M Karelson2, T Salum3, E Aunin1, P Reemann1,2, K
Abram2, E Vasar1, 4, H Silm2, and S Kõks1,4,5
1
University of Tartu, Estonia, Department of Physiology
2
University of Tartu, Estonia, Clinic of Dermatology
3
Tartu University Hospital, United Laboratories, Estonia
4
University of Tartu, Estonia, Centre of Translational Medicine
5
Estonian University of Life Sciences, Estonia, Institute of Veterinary Medicine
and Animal Sciences
Background/Aims: The corticotrophin-releasing hormone-proopiomelanocortin
(CRH-POMC) system coordinates stress response and melanogenesis in skin.
Potential role of this system in the pathogenesis of vitiligo is not clear. This
study analyzes the expression of genes related to the CRH-POMC system,
including melanin-concentrating hormone (PMCH), endogenous opioids and
their receptors.
Methods: With quantitative real-time-PCR the mRNA expression of twelve
genes were measured in samples extracted from skin biopsies of vitiligo patients
and healthy controls.
Results: The mRNA expression level of prepronociceptin (PNOC) and its
receptor opioid receptor like 1 (OPRL1) was higher in vitiligo involved skin
compared to healthy controls. The expression level of (PMCH) has increased in
uninvolved and involved vitiligo skin; melanin concentrating hormone receptor
1 (MCHR1) expression was higher in uninvolved and lower in involved vitiligo
skin compared to healthy control skin.
Conclusions: It was found that in addition to CRH-POMC system genes, the
expression of some genes from related systems (MCH and PNOC system) is
also altered in the skin of vitiligo patients and both MCH and endogenous
opioids derived from PNOC gene may participate in the pathogenesis of vitiligo.
Our study supports the focal role of CRH-POMC-MCH system in the skin in the
maintenance of skin homeostasis.
184
ABSTRACT 146
EFFECTS OF A HIGH SPONTANEOUS MUTATION RATE IN
MAMMALIAN GERMLINE BY USING MUTATOR MICE MODIFIED
REPLICATIVE DNA POLYMERASE DELTA
Arikuni Uchimura1, Yuko Hidaka1, Ikuo Miura2, Shigeharu Wakana2, and
Takeshi Yagi1
1
Graduate School of Frontier Biosciences, Osaka University, 2Technology and
Development Team for Mouse Phenotype Analysis, Japan Mouse Clinic, RIKEN
BioResource Center
Germline mutation rate is a very important parameter in mammalian genetics
and evolutionary biology. Arising frequent mutations cause an increase in
patients suffered from various congenital diseases and a decrease in mean fitness
among population. Although a spontaneous deleterious mutation rate is known
to be tremendously high in a mammalian germline, we know little about what
effect is caused by such a frequent mutation through several generations. To
investigate this, we established a new experimental model. In this model, we
generated a mutator mouse strain on a C57BL/6 background, in which the
spontaneous mutation rate was increased by modifying a replicative DNA
polymerase ƒÂ with a gene targeting method. The mutator mice were then bred
by mating between siblings for several generations, producing several mutation
accumulation mouse lines (MA lines). In these MA lines, after 4 years breeding
we found several phenotypic changes, a decrease in reproductive ability, an
increase in the number of mice suffered from severe hydrocephalous, and
sudden death without visible defects. In addition, we also found some
phenotypic variations and several heritable abnormal phenotypes in each MA
line. The heritable abnormal phenotypes included a short leg, reduced toe
number and a chirping behavior (of which mutants emitted clearly audible
sounds in the human hearing frequency range like a songbird). Our novel
experimental model could provide a clue to understand relationships between
the germline mutation rate and mammalian evolution and a risk of high mutation
rate for mammalian prosperity.
185
ABSTRACT 147
TOOLING UP MGI FOR A DELUGE IN MOUSE PHENOTYPE AND
DISEASE MODEL DATA
Anna Anagnostopoulos, Janan Eppig, and Mouse Genome Informatics Group
Global efforts to functionally annotate the mouse genome through large-scale
mutagenesis and phenotyping consortia have catapulted the size and diversity of
new mouse resources aimed at building predictive models of human disease.
The Mouse Genome Informatics resource (MGI, www.informatics.jax.org) for
the genetics, genomics and biology of the laboratory mouse grants free access to
current integrated biological knowledge spanning from sequence to phenotype
and disease model information to unified data on worldwide mouse resource
holdings via IMSR. MGI curates aberrant mouse phenotypes in the context of
mutations, strain variations, QTLs and complex traits shaped by intricate
epigenome-environment networks. To support the data deluge fueled by ongoing
translational research, MGI has devised automated processes to gradually
incorporate all multipurpose alleles generated by various IKMC partners, and
built a Recombinase Data Portal (www.creportal.org) to provide expression and
specificity data for each cre-containing transgene and knock-in allele, aiming to
integrate phenotype characterization of newly generated mutant mouse lines as
available. MGI users can access mouse phenotypes, alleles and disease models
of preclinical value using a suite of tools, including an enhanced Quick Search
Tool, Mouse GBrowse, web-based vocabulary browsers and data-specific query
forms. Robust search inputs include standardized terms from the Mammalian
Phenotype Ontology, a widely adopted ontological model that enables
phenotype annotations to background-specific allelic genotypes at varying
degrees of granularity. Use of human disease (OMIM) terms serves to harness
associations between phenodeviant mouse features and orthologous human gene
mutations or disease syndromes for which defined mouse genotypes model the
human condition. We will review MGI phenotype viewing options, customized
retrieval of complex phenogenomic datasets and disease model mining from
various perspectives. Supported by NIH grant HG000330
186
Appendix 1
BIOINFORMATICS WORKSHOPS
Co-Organizers:
Carol Bult, Ph.D. (The Jackson Laboratory, Bar Harbor, ME, USA)
Deanna Church, Ph.D. (National Center for Biotechnology Information,
Bethesda, MD, USA)
Special Guest Organizer for 2010:
Valerie Schneider, Ph.D. (National Center for Biotechnology Information,
Bethesda, MD, USA)
Tutorial 1
Room “Polymnia”
Getting the Most from the Mouse Reference Genome Assembly
Instructor:
Valerie Schneider, Ph.D. (National Center for Biotechnology Information,
Bethesda, Maryland, USA)
The mouse reference assembly is among the highest quality genomes ever
sequenced, and plays diverse roles in research. In 2007, the Genome Reference
Consortium (GRC) assumed responsibility for the mouse reference genome and
is engaged in ongoing efforts to update and improve this assembly. This
workshop is designed to provide attendees with the knowledge and tools needed
to make the most of this valuable and dynamic resource.
Topics covered in this workshop will include:
•
•
•
•
•
•
•
Creating the Mouse Genome Assembly
Sequencing Technologies and Assembly Methods
Assessing Assemblies
Assembly Management
Updates to the Reference Assembly
Reporting Problems with the Assembly
Tools for Accessing the Assembly
187
Tutorial 2
Room “Erato”
Mouse Phenome Database
Instructor:
Molly Bogue, Ph.D. (The Jackson Laboratory, Bar Harbor, Maine, USA)
The Mouse Phenome Database (MPD; phenome.jax.org), maintained at The
Jackson Laboratory, is the product of an international community effort to
collect quantitative phenotypic and genotypic data on laboratory mouse strains.
Since last year, MPD has added several comprehensive datasets, including
autism-relevant behaviors, toxicogenetic analysis of drug-induced liver injury,
susceptibility to alcohol intoxication, aging-related phenotypes, reproductive
parameters, cardiovascular responses to beta-adrenergic blockade or activation,
adiposity, and vestibular and eye parameters. MPD is also in the process of
integrating gene expression datasets, which are eligible for analysis with MPD
tools.
MPD is indispensable for helping researchers select optimal strains for many
research applications. MPD contains:
• Data for over 600 strains of mice (inbred, RI, consomic, etc.)
• Hundreds of baseline measurements of biomedically-relevant phenotypes
• A growing collection of data from treated mice, e.g., drugs; carcinogenic or
toxic compounds; high-fat diet
• Detailed protocols and environmental conditions of the test animals
• New SNP datasets (more strains and more genomic locations)
• Gene expression microarray data
• Pathology survey of aged inbred strains of mice
Topics covered in this workshop will include:
• Introduce the Mouse Phenome Project and MPD
• Find data sets related to specific areas of biology
• Find all data available in MPD for a particular strain
• Find details about a project submitted to MPD
• Perform basic statistical analysis on MPD data sets
• Combine phenotype measurement data with large-scale SNP data
• Compare SNPs across multiple strains
• Query SNPs based on MGI objects (genes, MIT markers, microRNAs,
and QTLs)
• Search for phenotype correlations across the entire MPD
• Create a “shopping cart” of user-defined data for custom analysis
• Download data from MPD
• Making suggestions to MPD
188
Tutorial 3
Room “Kalia”
A Tour of Mammalian Genome Annotation
Instructors:
Laurens Wilming, Ph.D. & Mark Thomas, Ph.D. (Havana group, Wellcome
Trust Sanger Institute, Hinxton, UK)
A reference gene set of accurately, detailed and comprehensively mapped loci
with associated features is crucial for genomic and genetic research. The
Wellcome Trust Sanger Institute is involved in producing reference gene sets in
human and mouse that includes VEGA, Ensembl and CCDS. The human
genome is annotated and analysed as part of the ENCODE (encyclopedia of
DNA elements) consortium. As part of the International Knock-out Mouse
Consortium (IKMC) we provide the annotation for the mouse knock-out
projects, together with collaborators at Washington University and the
University of Manitoba. We also lead the Mouse Genomes Project, sequencing
and analysing the genomes of 17 laboratory mouse strains that differ with
varying degrees from the reference C57BL/6J strain.
In this workshop we will present the following:
•
•
•
•
Explanation of annotation, biotypes and Ensembl-VEGA gene merges,
specifically in the context of the IKMC, ENCODE, CCDS and
community annotation projects
Accessing genome annotation using genome browsers (VEGA/Ensembl
and UCSC) and other web resources
Identification of knock-out mouse designs and associated resources
using genome browsers and the IKMC web portal
Walk-in clinic, help desk or Q&A: interactive sessions helping
delegates improve genome annotation and answer their annotation
questions.
Delegates are encouraged to bring us their annotation queries for discussion
during the “walk-in clinic” session or anytime during the conference or
afterwards.
189
PARTICIPANTS
59 45 101 60 42,147 22 61 62 37,63,133 64 65,66,67 107 86 19 28 68 30 6 58 32 69 70 71 56 36 99 98 7 72 73 90 74 Acosta, Deanna - Albert Einstein College of Medicine, USA
Adams, Niels - Mary Lyon Center, UK
Agami, Reuven - The Netherlands Cancer Institute, Netherlands
Aidinis, Vassilis - BSRC Alexander Fleming, Greece
Al-Mahdawi, Sahar - Brunel University, UK
Amato, Roberto - University of Naples Federico II, Italy
Anagnostopoulos, Anna - The Jackson Laboratory, USA
Angel, Joe - The University of Texas, USA
Arkell, Ruth - Early Mammalian Development, Australia
Ball, Simon - Medical Research Council, UK
Balling , Rudi - Luxembourg Center for Systems Biomedicine, Luxembourg
Bauer, Beth - University of Missouri, USA
Beier, David - Harvard Medical School, USA
Benavides, Fernando - M.D. Anderson Cancer Center, USA
Bergstrom, David - The Jackson Laboratory, USA
Birling, Marie-Christine - ICS, France
Boivin, Gregory - McGill University, Canada
Brodnicki, Thomas - St. Vincents Institute, Australia
Brown, Steve - Medical Research Council (MRC), UK
Bryda, Elizabeth - University of Missouri, USA
Bucan, Maja - Univ. of Pennsylvania, USA
Cabin, Deborah - McLaughlin Research Institute, USA
Calaway, John - University of North Carolina, USA
Carlson, George - McLaughlin Research Institute, USA
Charizopoulou, Nikoletta - NIDCD/NIH, USA
Chen, Yen-Hui - Taiwan Mouse Clinic, Taiwan
Clare, Simon - Sanger Institute, UK
Connolly, Diana - Albert Einstein College of Med. of Yeshiva University, USA
Copeland, Neal - Inst. Of Molecular and Cell Biology, Singapore
Croniger, Colleen - Case Western Reserve University, USA
Cross, Sally - MRC Human Genetics Unit, UK
Danecek, Petr - Wellcome Trust Sanger Institute, UK
Das, Sabyasachi - Emory University, USA
de la Casa Esperon, Elena - University of Castilla-La Mancha, ES
Didion, John - Univ. of North Carolina, USA
Doerner, Stephanie - Case Western Reserve University, USA
Donahue, Leah Rae - The Jackson Laboratory, USA
Douni, Eleni - BSRC Alexander Fleming, Greece
Eaton, Sally - Medical Research Council, UK
190
42 76 47 75 53 77 52 78 26 82 79 48 23 57 100 39 55 13 9 87 89 92 97 16 96 15 3 91 93 Economides, Aris - Regeneron Pharmaceuticals Inc., USA
Ekker, Marc - University of Ottawa, Canada
Elliott, Rosemary - Roswell Park Cancer Inst, USA
Engel, Nora - Temple University School of Medicine, USA
Estabel, Jeanne - Wellcome Trust Sanger Institute, UK
Foote, Simon - Menzies Research Inst., Australia
Forejt, Jiri - Instututre of Molecular Genetics AS CR, CZ
Fukumura, Ryutaro - RIKEN BRC, Japan
Galas, David - Inst. For Systems Bio, USA
Gerdin, Anna-Karin - Wellcome Trust Sanger Institute, UK
Goggolidou, Paraskevi - Medical Research Council, UK
Gondo, Yoichi - RIKEN, Japan
Gunn, Teresa - McLaughlin Research Institute, USA
Hagn, Michael - Helmholtz Zentrum München, Germany
Hassan, Ahmad - Medical Research Council (MRC), UK
Heaney, Jason - Case Wester Reserve University, USA
Hentges, Kathryn - The University of Manchester, UK
Herman, Gail - The Ohio State University, USA
Horvat, Simon - University of Ljubljana, Animal Science, Slovenia
Houri-Haddad, Yael - Boston University, USA
Hrabe de Angelis, Martin - Helmholtz Zentrum München, Germany
Humphrey, Neil - The Univ. of Manchester, UK
Hunter, Kent - National Cancer Institute, USA
Ioakeimidis, Fotios - BSRC "Alexander Fleming", Greece
Iraqi, Fuad - Tel Aviv University, Israel
Ishida, Saeko - Kyoto University, Japan
Ivanov, Andrey - GEOKHI RAS, Russia
Jackson, Ian - MRC Human Genetics Unit, UK
Jaubert, Jean - Institut Pasteur, France
Jenkins, Nancy - Indt. Of Molecular and Cell Biology, Singapore
Johansen, Jeanette - Karolinska Institutet, Sweden
Kaerst, Stefan - Humboldt-Universität zu Berlin, Germany
Kane, Leanne - Wellcome Trust Sanger Inst., UK
Karalis, Katia - BRFAA Greece and Childrens Hospital, Greece
Keane, Thomas - Wellcome Trust Sanger Institute, UK
Keegan, Catherine - Pediatrics and Human Genetics, USA
Kember, Rachel - SGDP, UK
Khrameeva, Ekaterina - Moscow State University, Russia
Koide, Tsuyoshi - Mouse Genomics Resource Laboratory, Japan
Kong, Philip - Kennedy Institute, UK
Korthaus, Dirk - Takeda SingaporePte Ltd., Singapore
191
33 83 43 95 102 118 119 14 121 18 85 54 110 112 117 17 4 111 35105 106 11 115 104 51 24 46 94 108,109 38 114 145 116 Kuramoto, Takashi - Kyoto University, Japan
Lad, Heena - MRC Harwell, UK
Leake, John - Rady Children's Hospital San Diego, USA
LeBoeuf, Renee - University of Washington, USA
Lewis, Morag - Wellcome Trust Sanger Institute, UK
Libert, Claude - Ghent University, Belgium
Lilleoja, Rutt - University of Tartu, EE
Lindfors, Charlotte - Molecular Medicine and Surgery, Sweden
Lo , Chiao-Ling - Purdue University, USA
Logan, Darren - Wellcome Trust Sanger Institute, UK
Lossie, Amy - Purdue University, USA
Lucas, Mark - Sanger Institute, UK
Maddatu, Terry - The Jackson Laboratory, USA
Marton, Jennifer - McGill University, Canada
Mashimo, Tomoji - Kyoto University, Japan
Masuya, Hiroshi - RIKEN, Japan
Matin, Angabin - MD Anderson Cancer Center, USA
Mburu, Philomena - MRC Harwell, UK
Mekada, Kazuyuki - RIKEN BioResource Center, Japan
Miller, Darla - UNC at Chapel Hill, USA
Mock, Beverly - NCI, NIH, USA
Mohammadi, Amir - The Australian National University, Australia
Montagutelli, Xavier - Institut Pasteur, France
Murata, Takuya - RIKEN BRC, Japan
Murray, Stephen - The Jackson Laboratory, USA
Nadeau, Joseph - Inst. For Systems Bio, USA
Neuhold, Lisa - National Eye Inst./NIH/HHS, USA
Ning , Li - BGI Shenzhen, China
Niti, Alexandra - BSRC Alexander Fleming, Greece
Noben-Trauth, Konrad - National Institutes of Health, USA
Oota, Satoshi - RIKEN, Japan
Osawa, Gail - University of Michigan, USA
Pardo Manuel De Villena, Fernando - Univ. of North Carolina, USA
Parsons, Michael - MRC Harwell, UK
Peters, Jo - MRC Harwell, UK
Pook, Mark - Brunel University, UK
Potter, Paul - Medical Research Council, UK
Ramirez-Solis, Ramiro - Wellcome Trust Sanger Insti, UK
Reilly, Karlyne - NCI-Frederick, USA
Reimann, Ene - Tartu, Estonia, Estonia
Reinholdt, Laura - The Jackson Laboratory, USA
192
113 120 122,123 1 138 40 130 5 132 21 125 8 12 124 126 31 49 127 84 34 27 146 10 139 25 44 136 20 135 2 Ripoll, Vera - Medical Research Council, UK
Rowe, Lucy - The Jackson Laboratory, USA
Ryder, Edward - Sanger Institute, UK
Schneider, Valerie - DHHS/NIH/NLM/NCBI, USA
Schughart, Klaus - Helmholtz Centre for Infection Research, Germany
shusterman, ariel - Department of Prosthodontics, IL
Simon, Michelle - Medical Research Council, UK
Skarnes, William - The Wellcome Trust Sanger, UK
Slater, Guy - Wellcome Trust Sanger Institute, UK
Smith, Rebecca - Kings College London, Institute of Psychiatry, UK
Snaith, Mike - Medimmune Ltd, UK
Snoek, Margriet - Netherlands Cancer Institute, NL
Sorg, Tania - ICS, France
Spiezio, Sabrina - Case Western Reserve Univ., USA
Stamova, Boryana - University of California, USA
Steel, Karen - Wellcome Trust Sanger Institute, UK
Stephen, Louise - University of Manchester, UK
Su, Zhaoliang - China
Sunter, David - Wellcome Trust Sanger Institute, UK
Suzuki, Tomohiro - RIKEN, Japan
Svenson, Karen - The Jackson Laboratory, USA
Szalai, Gabor - University of South Carolina, USA
Takada, Toyoyuki - National Institute of Genetics, Japan
Tanaka, Nobuhiko - RIKEN BRC, Japan
Tateossian, Hilda - MRC Harwell, UK
Thomas, Mark - Wellcome Trust Sanger Institute, UK
Threadgill, David - North Carolina State Univ., USA
Tinsley, Louise - Medical Research Council (MRC), UK
Traka, Maria - The University of Chicago, USA
Uchimura, Arikuni - Osaka University, Japan
Van Schaick, Jessica - The George Washington University, USA
Vancollie, Valerie - Wellcome Trust Sanger Inst., UK
Vlangos, Christopher - University of Michigan, USA
WAKANA, Shigeharu - RIKEN BRC, Japan
Wakeland, Edward - UT Southwestern Medical Center, USA
Wallace, Marsha - Cornell University, USA
Weiss, Ervin - The Hebrew University, Israel
Westrick, Randal - University of Michigan, USA
White, Jacqul - Wellcome Trust Sanger Inst., UK
Wilming, Laurens - Wellcome Trust Sanger Institute, UK
Wiltshire, Sean - McGill University, Canada
193
29,140 143 134 131 80,81 129 128 50 Wiltshire, Tim - University of North Carolina, USA
Wong, Kim - Wellcome Trust Sanger Institute, UK
Wu, Jiaqian - Stanford University, USA
Xuengang, Wang - BGI Shenzhen, China
Yagi, Takeshi - Osaka University, Japan
Yang-Yen, Hsin-Fang - Academia Sinica, Taiwan
Yann, Herault - ICS/IGBMC-TAAM, France
Yen, Jeffrey - Institute of Biomedical Sciences, Taiwan
Yu, Eugene - Roswell Park Cancer Institute, USA
Yuan, Rong - The Jackson Laboratory, USA
194
SPONSORS
Aegean Conferences and the IMGS wish to gratefully acknowledge the
following sponsors for their generous support of this meeting.
Funding for student scholarships was made possible by:
2R13HG002394 from the following ICs at NIH: NHGRI,
NIMH, NICHD, NIAID, NIEHS, NCRR, AND NINDS
BGI SHENZHEN
Cambridge Journals
Genomics
Genesis
Genetics Society of America
Genome Research
Mammalian Genome
The Ellison Medical Foundation
195
UPCOMING EVENTS
4th International Conference on Tissue Engineering
Minoa Palace Conference Center, May 27-June 1, 2011
6th Leukocyte Signal Transduction Workshop
Minoa Palace Conference Center, June 1-5, 2011
9th International Conference Pathways, Networks, and Systems Medicine
8th International Conference Innate Immunity
5th International Workshop on Complement Associated Diseases, Animal
Models, and Therapeutics
Sheraton Conference Center, Rhodes, Greece June 22-27, 2011
3rd International Summit on Hurricanes and Climate Change
Sheraton Conference Center, Ixia, Rhodes, Greece June 27-July 2, 2011
4th International Conference on Crossroads between Innate and Adaptive
Immunity
Royal Myconian Hotel. Chania, Crete, Greece, Sept. 11-16, 2011
1st International Conference on Immunometabolism: Molecular and
Cellular Immunology of Metabolism
Aldemar Knossos Royal Village, Crete, Greece, Sept 18-23, 2011
5th International Conference on Autoimmunity: Mechanisms and Novel
Treatments
Aldemar Knossos Royal Village, Crete, Greece, Sept 18-23, 2011
196
CONFERENCE CENTER MAP
197
ALDEMAR HOTEL MAP
198
Aegean Conference Proceedings
199
To purchase using the Aegean Conferences discount download the form:
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200

Abstracts - International Mammalian Genome Society

Transcription

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