Stressökologie (SOE 1) - Der WWW2

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Stressökologie (SOE 1) - Der WWW2
Fachkurs für das Hauptstudium
Stressökologie (SOE 1)
Molekularbiologie, Genetik und
Informatik mit C. elegans
09.– 19. Januar 2007
Dr. Ralph Menzel
Institut für Biologie - Gewässerökologie
HUMBOLDT UNIVERSITÄT zu BERLIN
Arboretum, Späthstr.80/81, 12437 Berlin
2
CONTENTS
Page
Contents
2
Eckdaten für die Protokolle (in German)
3
Schedule
4
Talks
5
Sicherheitsbestimmungen (in German)
6
Module 1
Manipulating worms, recognizing stages, life cycle
7
Module 2
Demonstration test-crosses
10
Module 3
Recognizing standard mutant phenotypes
11
Module 4
Decontaminating cultures by bleaching
13
Module 5
Freezing worms for long-term storage
14
Module 6
Examining worms by Nomarski DIC microscopy
15
Module 7
Use of Green Fluorescent Protein reporters
16
Module 8
Gene inactivation by RNAi (bacterial feeding)
17
Module 9
Extraction of RNA from worms
19
Module 10
Temperature shifts on temperature-sensitive mutants
21
Module 11
lacZ staining of reporter transgene strains
22
Module 12
Ballistic transformation of C .elegans (in German)
23
Module 13
Single worm PCR (in German)
25
Module 14
Reproduction and thermo-tolerance (in German)
27
Module 15
Informatics resources
29
References
31
3
ECKDATEN FÜR DIE PROTOKOLLE
• Es muss von jedem Studenten ein individuelles Protokoll angefertigt werden.
• Gliederung: Modulbezogen mit jeweils kurzer Einleitung, Ergebnisse und Diskussion.
• Letzter Abgabetermin:
5.03.
15°°Uhr
8.03.
9-15°°Uhr
12.03.
15°° Uhr
• Abholung der Protokolle für möglicherweise
notwendige Verbesserungen:
• Letzte endgültige Abgabe /Scheinausgabe:
Keine Scheinvergabe mehr nach den genannten Terminen!
SCHEDULE
Day
Time
9.00
Tuesday09/01
Wednesday, 10/01
Thursday, 11/01
Friday, 12/01
Day 1
Day 2
Day 3
Introduction
(Belehrung)
Lecture
8, 1
Sa
Su
Mo
Tuesday, 16/01
Wednesday, 17/01
Thursday, 18/01
Friday, 19/01
Day 4
Day 8
Day 9
Day 10
Day 11
2 student talks
2 student talks
13
2, 4, 5
2 student talks
Lecture
Lecture
Guest Lecture
Nadine Saul
Kerstin Pietsch
Birgit Gerisch
12, 14
1, 2
1, 2
2, 4, 5
Ralph Menzel
Lecture
10.00
1
11.00
12
8, 14
6
7
2
4, 5
9
10
15
12.00
12
3
6
7
8
10
9
11
15 2, 4, 5, 11
13.00
Lunch
Lunch
Lunch
Lunch
Lunch
Lunch
Lunch
14.00
12
8
7
6
14
14
10
14
11
15.00
12
9
7
6
14
14
12
15
14
13, 10
16.00
1
9
14
12
15
12
17.00
7, 8
18.00
14
Christian Steinberg
Lunch
1, 3
13
Summation
and Clean-up
5
TALKS
Always at 9 am in the seminar room (3th floor)
Tuesday (09/01)
Dr. Ralph Menzel
"The nematode C. elegans as model organisms - Introduction to
the course"
Wednesday (10/01) Prof. Dr. Christian Steinberg (10 am !)
"Humic substances as ecological driving forces in inland water"
Thursday (11/01)
Nadine Saul
"The phenomenon of hormesis"
Friday (12/01)
Kerstin Pietsch
"Blueberry polyphenols increase lifespan and thermo-tolerance in
C. elegans"
Tuesday (16/01)
Dr. Birgit Gerisch, MPI für Molekulare Genetik, Berlin
“Aging in C. elegans”
Student talks:
-
on January 17th, 18th, (at 9 am) and 19th. (at 10 am)
assignment of topics during the course meeting on December 18th
20 min talk, 10 min discussion
MS Powerpoint presentation
6
SICHERHEITSBESTIMMUNGEN
Zum Umgang mit potentiell gefährlichen Stoffen
Im Kurs wird mit einer Reihe potentiell gefährlicher Stoffe umgegangen, deren
Handhabung entsprechende Vorsichtsmaßnahmen erfordert.
•
potentiell karzinogene Stoffe:
Ethidiumbromid (DNA Farbstoff in Agarosegelen, Auszug aus dem Datensicherheitsblatt
hängt im Labor aus)
•
potentiell gefährliche Chemikalien:
β-Mercaptoethanol (reduzierend, giftig, SH-Gruppen Schutz), alle Arbeiten unter dem
Abzug, Handschuhe verwenden
Natriumhypochloritlösung (12% Cl → ätzend), Handschuhe und Schutzbrille verwenden !
Chloroform bzw. Trizol (enthält das Lösungsmittel Phenol), alle Arbeiten unter dem Abzug,
Handschuhe verwenden
Ethanol (leicht brennbar, Lösungsmittel).
Grundregeln für biochemische und gentechnische Arbeiten:
•
•
•
•
•
•
•
•
•
•
Gentechnische Arbeiten dürfen nur in den Räumen durchgeführt werden, die zur
genehmigten gentechnischen Anlage gehören.
Der Arbeitsplatz sollte möglichst aufgeräumt sein und sauber gehalten werden.
Am Arbeitsplatz darf grundsätzlich nicht gegessen, geraucht oder getrunken
werden.
Im Labor ist personenbezogene Schutzbekleidung (Kittel, bei Bedarf Handschuhe
bzw. Mundschutz) zu tragen. Vor Verlassen der Laborräume ist die Schutzbekleidung abzulegen. Die Arbeitskleidung ist getrennt von normaler Kleidung
aufzubewahren
Mundpipetieren ist grundsätzlich untersagt. Es sind vorhandene Pipentierhilfen zu
verwenden.
Türen und Fenster der Arbeitsräume sind während der Arbeiten verschlossen zu
halten.
Das für die Arbeiten nicht mehr benötigte biologische Material wird in den dafür
vorgesehen Behältnissen gesammelt und durch mindestens 20 min autoklavieren
bei 121°C unschädlich gemacht. Kontaminierte Geräte sowie benutztes Einwegmaterial wird in gleicher Weise behandelt.
Während einer Schwangerschaft dürfen keine gentechnischen Arbeiten durchgeführt werden!
Mit Lösungsmitteln bzw. β-Mercaptoethanol ist unter einem Abzug zu arbeiten.
Mögliche Verletzungen müssen umgehend dem Kursleiter gemeldet werden.
Zur Vorsorge immer bei den Betreuern nachfragen und sich Anweisungen holen.
7
Module 1 Manipulating worms, recognizing stages and sexes, life
cycle
Worms are normally grown on NGM agar, using OP50, a uracil-requiring
strain of E. coli as a food source. NGM plates are inoculated with OP50 and incubated
at 37°C for 1 day, to create a bacterial lawn. Worms can then be added to the lawn
and grown. In a few days, they will eat up all the food and begin to starve. A starved
stock plate can be kept for 8 weeks or more at 15°C, without losing viability in the
worm population. The main limitation is desiccation: the agar medium eventually dries
out completely (“potato chip” state) and the worms die. Sealing the plate with parafilm
will delay this.
Worms can be transferred to a fresh culture plate on an individual basis,
using a worm pick, or by loop transfer (using a standard bacteriological inoculating
loop to scoop up a small amount of worm-infested agar), or by chunk transfer (using a
flamed scalpel blade to cut out a chunk of the starved plate). Transferring a single
starved worm by means of a pick is not always reliable, so the latter two methods are
both more reliable and easier.
Individual worms are usually manipulated using a worm pick. This is a
short length of platinum wire (we use 0.3 mm diameter), mounted in a glass handle,
with the end flattened into a curved scoop. The pick should always be flamed in a gas
or alcohol burner before use, in order to ensure sterility. Let the pick cool before
touching a worm with it, either in air for a few seconds, or by touching the pick against
the agar surface of the plate. A worm can be picked up by using the pick like a spoon.
When doing this, try not to break the surface of the agar -- if there are many breaks or
gouges in the surface, the worms will burrow into the agar, rather than living on the top
surface. Once a worm has been picked, it will begin to dry out, so place it on its
destination plate within 30 seconds or so.
An alternative and usually superior procedure is to scoop up a gob of
sticky bacteria on the end of the platinum wire, and pick worms by adhesion to the
sticky gob. This has the advantage of reducing the risk of damaging either worms or
agar surface, and it also allows one to pick up several worms at once, all adhering to
the sticky gob. Keep a small plate of old OP50 as a source of sticky bacteria
(supplied).
Recognizing stages and sexes: L1 to L3 larvae cannot easily be sexed by
inspection with a dissecting microscope, and the stages look superficially similar,
except in size. At L4 stage, both males and hermaphrodites have a distinctive marbled
appearance. At this stage, males have a swollen white patch at the tail end of the
body, where the male copulatory organs are developing. Hermaphrodites have a small
white patch in the middle of the body, where the vulva is developing. At the adult
stage, sexes are more easily distinguished: hermaphrodites are longer and fatter, with
a visible vulva and eggs developing inside the uterus. Males are thinner and have a
modified tail, which looks like a hook at low power. Adult males are also behaviorally
recognizable, constantly sliding over hermaphrodites in mating attempts.
At the end of each larval stage, worms enter lethargus, a period of
inactivity when they undergo molting. The lethargus at the end of L4 is a convenient
synchronization point. Dauer larvae, which are executing the alternative third stage of
development, have a thin, dark, whip-like appearance. They sometimes accumulate in
8
water droplets on the lids of culture plates. Unlike all other stages except the egg, they
are resistant to harsh treatments such as washing in 1% SDS, which kills both adults
and non-dauer larvae, but leaves dauers unharmed.
Recognizing sexes and stages
Day 1
1. Spreading plates. You will be supplied with a suspension of E. coli OP50, grown to
saturation in nutrient broth. Drop or spread about 10 µl of this suspension on each
small NGM plate, using a 1ml pipette. Try not to scratch the agar surface, or to
spread the bacteria too close to the edge of the plate. Spreading the lawn in loops
or pretzel shapes creates lots of edges, which the worms like. The lawn will be
dense enough to use after overnight incubation at room temperature, or a few
hours at 37°C.
2. Practice picking up worms and transferring them to fresh plates. You will be
supplied with some pre-spread plates, and plates of a wild type hermaphrodite
culture, and a wild type male/hermaphrodite culture.
3. From wild type male plate, pick to three separate small plates:
A.
10 adult hermaphrodites
B.
10 L4 hermaphrodites
C.
10 adult males
Incubate at 25°C.
Day 2
4. Check plates: On plate A there should be many eggs and hatchling larvae. On
plate B there should be a few eggs. On plate C, if you have correctly picked only
males, there will be no eggs or larvae present. Probably some or all of the males
will have tried to swim up the side of the plate and will be visible on the plastic wall
as desiccated corpses. Males tend to do this if there are no hermaphrodites
around.
The lifecycle
Day 1
1. Pick two single eggs from the hermaphrodite culture to a small plate, incubate at
25°C.
Day 2
2. Examine these plates, observe L1/L2 larvae. Both eggs should have hatched.
Day 3
3. Observe L3/L4 larva.
Day 4
4. Observe egg-laying adults, first progeny larvae hatching
Day 8
5. Observe progeny larvae developing, exponential increase in population with next
generation, leading to eventual consumption of all bacterial food, and a starving
worm population.
9
Note that the clear patch observed in L4 animals differs from that seen in L2 and L3
animals. Also, L4 animals are clearly larger in size than L2 and L3 animals.
10
Module 2 Demonstration test-crosses
Two demonstration crosses will be carried out, one involving two unlinked
recessive mutations (A), the other involving two linked recessive mutations (B).
Cross A: This uses a dumpy mutation on chromosome V (dpy-11) and an
uncoordinated (unc-7) mutation on the X chromosome. Double mutant hermaphrodites
(dpy-11; unc-7, Dumpy and Uncoordinated in phenotype) will be crossed with wild type
males (P0). This will give rise to F1 heterozygous hermaphrodite progeny (dpy-11/+;
unc-7/+), which will be phenotypically wild type (WT), and to male progeny which will
be Uncoordinated but non-Dpy (dpy-11/+; unc-7/O), because the unc-7 mutation is
sex-linked. The F1 hermaphrodite progeny will be selfed, and the two phenotypes Dpy
and Unc will be observed to segregate in the F2 generation, in a Mendelian ratio of 9
WT : 3 Dpy : 3 Unc : 1 Dpy Unc.
Cross B: This uses the same dpy-11 mutation on chromosome V, and an unc
mutation on the same chromosome (unc-42), located about 2 centiMorgans (2%
recombination) away. Double mutant hermaphrodites (dpy-11 unc-42 , Dpy Unc in
phenotype) will be crossed with wild type males (P0). The F1 cross-progeny will be WT
in phenotype, both hermaphrodites and males (dpy-11 unc-42/+ +). The F1
hermaphrodites will be selfed, and the F2 progeny examined: these will be mostly WT
and Dpy Unc, with a few rare Dpy non-Unc and Unc non-Dpy recombinants.
Day 4
1.
For each cross, place 3 adult hermaphrodites (Dpy Unc) on a small plate and add
6 WT adult males. Incubate at 25°C.
Day 8
2.
Examine crosses: for cross A, observe WT hermaphrodite progeny and Unc male
progeny. For cross B, observe WT hermaphrodite and WT male progeny. From
each cross, pick a single young adult hermaphrodite to a separate small plate.
Incubate at 25°C.
Day 9 and 10
3.
Transfer each hermaphrodite to a fresh plate, so that only one day’s worth of
eggs is laid on each plate. This makes the population more synchronous and
easier to score.
Day 11
4.
Examine F2 progeny. For cross A, observe independent segregation of Dpy and
Unc phenotypes. For cross B, observe that most animals are either WT or doubly
mutant, DpyUnc. Rare recombinants (Dpy non-Unc or Unc non-Dpy) will be
visible as a few percent of the population.
Reference: Brenner (1974)
11
Module 3 Recognizing standard mutant phenotypes
This module demonstrates some of the common mutant phenotypes that
are used in C. elegans genetics. Some are easy to recognize, on the basis of gross
morphology. Others can only be recognized at certain stages, or by simple behavioral
testing.
Day 2 and onward
Each pair will be provided with stock plates for 10 commonly used mutants, and two
wild-type strains. Examine by dissecting microscope. Tap the plate, or prod the worms
with a worm-pick, in order to elicit responses and test for reverse and forward
movement.
Mutants:
1.
dpy-10, allele e128. “Dumpy”: animals are much shorter than wild-type, at all
stages. There are about 25 other dumpy genes so far defined in C. elegans,
which have similar but not identical phenotypes. This gene, like some but not all
of the other dpy’s, encodes a cuticle collagen.
2.
rol-1, allele e91. “Roller”: adult animals roll about their long axes as they
move, and as a result tend to move in circles. Note that larvae do not roll. This
mutant is left-handed roller, because it rotates in a anti-clockwise direction. The
rolling arises from a helical twist imparted to the cuticle by a defective collagen.
This roller mutation is recessive. Certain other roller mutations are dominant,
and one of these is used as a standard transformation marker.
3.
bli-2, allele e768. “Blister”: adult animals develop fluid filled blisters on the
body surface, resulting from accumulation of fluid in the space between the two
layers of the adult cuticle. Larvae are not blistered, because they have a singlelayered cuticle. This gene, like dpy-10 and rol-1, encodes a collagen, probably a
component of the struts separating the layers of the adult cuticle.
4.
lon-2, allele e678. “Long”: animals are about 50% longer than wild-type. This
gene encodes a growth-factor related molecule.
5.
sma-2, allele e502. “Small”: animals are shorter and thinner than wild-type, but
do not have the fat appearance of Dpy mutants. sma-2 males also have
distinctive defects in the development of the tail (not visible in this
hermaphrodite stock). sma-2 encodes a Smad family member, involved in
TGFbeta signaling.
6.
unc-17, allele e245. “Uncoordinated, Coiler”: animals are unable to move
well, and spend most of their time curled up. They are able to lay eggs,
however. Mutants are resistant to cholinesterase inhibitors such as aldicarb,
and in fact move much better in the presence of such drugs. unc-17 encodes
the transporter molecule that loads acetylcholine into synaptic vesicles. It forms
part of a compound gene together with cha-1, which encodes the synthetic
enzyme choline acetyltransferase. Strong cha-1 mutants are unable to make
acetylcholine, and die as abnormal L1 larvae.
12
7.
lin-1, allele e1777. “Lineage defect, Muv (Multi-vulva)”: adult hermaphrodites
have up to six ventral protrusions, or pseudo-vulvae, resulting from vulval
divisions by all six P3.p -P6.p vulval precursor cells. lin-1 encodes a putative
transcription factor, belonging to the ETS family. It is regulated by a ras protein
kinase cascade, responding to extracellular signaling by the LIN-3 signal,
received by the LET-23 receptor tyrosine kinase.
8.
mec-3, allele e1338. “Mechanosensory defect”: animals are defective in the
response to light touch. This is a subtle phenotype, assayed by stroking worms
with a fine hair (e.g. eyebrow hair mounted on a toothpick, as supplied). Wild
type animals will respond to touch on the anterior body by reversing, and to
touch on the posterior body by going forward. Mec animals fail to respond to
light touch, and are generally lethargic, but will respond to a more vigorous
stimulus, such as a prod from a wire pick. mec-3 encodes a LIM class
homeoprotein, required for the proper differentiation of the six touch receptor
neurons.
9.
him-8, allele e1489. “High Incidence of Males”: populations contain many
males as well as hermaphrodites, because of X chromosome loss in
hermaphrodite gametogenesis. WT hermaphrodites produce only 0.2 % XO
male progeny, whereas him-8 hermaphrodites produce about 38% XO, as well
as 6% XXX hermaphrodites (these are shorter than normal XX hermaphrodites).
him mutants provide a useful source of males. him-8 encodes a novel protein.
10.
Wild-type strain: N2. This is the wild-type Bristol strain, originally isolated from
an English mushroom farm in the 1950’s. Almost all C. elegans is based on
derivatives of this original strain.
13
Module 4 Decontaminating cultures by bleaching
(Alkaline hypochlorite treatment)
Alkaline hypochlorite or Bleaching Solution (BS2X) conveniently
dissolves all worm tissues except eggs, which are largely resistant, and will also
destroy almost all bacterial and fungal contaminants, with the exception of certain
resistant spores. Bleaching is used to decontaminate cultures (this module) and also in
preparing bulk preparations of pure eggs.
Day 8
1. Place 5 µl of bleach (sodium hypochlorite, 12 - 15% available chlorine) and 5 µl of
2 N NaOH on a small seeded NGM plate, between the bacterial lawn and the edge
of the plate. From the contaminated plate provided, pick 6 - 10 gravid
hermaphrodites (adults containing lots of eggs) and deposit them in the drop of
solution. Incubate at room temperature.
Day 9
2. Inspect the cleaning plate: the picked worms should have completely dissolved,
leaving only cellular debris. The resistant eggs should have hatched, and the
resulting larvae will have crawled over to the bacterial lawn and begun feeding and
developing. Some resistant bacterial spores may have survived bleaching, so take
a scalpel, flame it and cut out the region of the plate including the bleaching spot.
Also, move some worms to a fresh plate.
Day 10 and 11
3. Inspect the cleaning plates; compare the decontaminated population with the
original contaminated population.
------------------------------------------------------------------------------Bleaching solution
For 50 ml:
Sodium hypochlorite (bleach)
(12 - 15% available chlorine; < 6 months old)
2 N sodium hydroxide
H2O
20 ml
25 ml
5 ml
14
Module 5 Freezing and thawing worms for long-term storage
Wild type and mutant strains of C. elegans can be stored frozen at –70°C
or in liquid nitrogen. They remain viable indefinitely in this state. The freezing protocol
involves slow cooling (about 1 degree per minute) in a 15 % glycerol solution. In order
to revive a frozen sample, it is warmed to room temperature rapidly, and spread on a
large NGM plate, in order to dilute the glycerol, which is somewhat toxic to the worms.
A thawed sample cannot be refrozen. The fraction of the population that survives
freezing depends on the state of the culture and sometimes on the genotype, but
under optimal conditions over 90 % survive. Starved cultures with lots of larval worms
(but not dauers) are the best material for freezing.
Day 8
1. Label one 1.5 ml freezing vials. Wash off a plate of starving N2 (wild type) worms
with about 1.5 ml M9 buffer. Take 0.75 ml of this suspension and add 0.75 ml FS
(freezing solution). Mix by vortexing and transfer into the freezing vial. Put it in a
styrofoam block and place the block in a -70° freezer. The Styrofoam provides
sufficient insulation to permit slow cooling (the exact rate is not critical). After six
hours or more, the tubes can be transferred to a liquid nitrogen tank, if available.
Day 9
2. Check viability: take the frozen tube and thaw by rolling between the palms of your
hands. When the sample has partly or completely melted, empty the contents of
the tube onto a large spread NGM plate. Examine the plate by dissecting scope,
while the liquid sample soaks into the agar. Notice that the worms appear initially
very crumpled, shrunken and immobile. After a few minutes, they will begin to
recover normal morphology, and can be seen to twitch and begin to swim.
Day 10 and 11
3. Check thaw plate; estimate what percentage of the sample survived freezing.
------------------------------------------------------------------Freezing solution (FS), for 100 ml)
NaCl
KH2PO4
Glycerol
NaOH (1 M)
Add H2O to:
0.585 g
0.68 g
30 g
0.56 ml
100 ml
Autoclave, then sterilely add 0.3 ml MgSO4, 0.1M.
15
Module 6 Examining worms by Nomarski DIC microscopy
Living worms can be conveniently examined by Nomarski differential
interference contrast microscopy, which permits visualization of all nuclei and many
other anatomical features. Worms are mounted on an agar or agarose pad, under a
coverslip. Worms will continue to swim in these conditions, so they are anaesthetized
for detailed examination.
Day 3
1. Making agarose pads: Take a drop of molten 3 % agarose (in M9 buffer) and
place it on a clean glass microscope slide, placed between two other slides
thickened with tape. Place another glass slide on the drop, at right angles to the
first, and press down to spread the agarose into a thin disc, 1 - 2 cm in diameter
(see Figure below). Wait a minute or so to for the agarose to set and then remove
the top slide by sliding laterally.
2. Mounting worms: Place a small drop (5 - 10 µl) of 20 mM sodium azide on the
agarose. Pick up 1 - 10 worms from a culture plate, using a wire worm-pick, and
place in the drop of buffer. The worms should be visible in the drop, thrashing
about vigorously. Carefully place a cover slip on top, trying not to create airbubbles.
3. Examine by Nomarski: Locate worms at low power (4.5 x). A 40x dry objective is
convenient for seeing many details of the anatomy. For more detailed examination,
a 63x or 100x objective is used, which requires oil immersion.
-------------------------------------------------------------------------------------------------------------------
16
Module 7 Use of Green Fluorescent Protein reporters
Green Fluorescent Protein provides an extremely useful in vivo reporter
for C. elegans. Some reporters are sufficiently bright that they can be seen and scored
using an epifluorescence dissecting microscope. However, much more detailed
examination is possible using a compound microscope fitted with fluorescence optics.
Day 1
1. Transfer worms of the provided strains by chunk transfer to a fresh seeded NGM
plate - use a flamed scalpel blade to cut out a chunk of the starved plate. Incubate
the plates at 25°C.
Day 3
2. Examine plates of worms using fluorescence compound microscope (use only the
4.5x and 10x magnification). Compare image with transmitted light, blue light, or
both. Five strains are provided:
Strain GFPA:
Almost all nuclei, apart from those in the germ-line, are
brightly fluorescent. These animals carry an extrachromosomal array expressing a sur5::GFP fusion, which is ubiquitously expressed and carries a strong nuclear
localization signal. Intestinal nuclei are especially bright because they are large and
polyploid. Occasional animals are non-fluorescent, because they have lost the
transgene array. Rare animals are genetic mosaics, with some fluorescent nuclei and
some non-fluorescent nuclei, because they have lost the array in part of the cell
lineage.
Strain GFP D: (DP132) These animals are fluorescent in all neurons as result of an
inserted transgene unc-19::GFP. The animals roll because the transgene was coinjected with the rol-6 transformation marker.
Strain GFP E: (PD4792) Strong fluorescence in pharyngeal muscle, weak
fluorescence in some other tissues. Carries mIs11, a mixed transgene array, which
includes myo-1::GFP (pharyngeal myosin)
Strain GFP F:
These animals have fluorescence in all body wall muscle
nuclei. This carries a transgene expressing myo-3::GFP with a nuclear localization
signal. Myo-3 is a body wall muscle myosin gene.
Strain P450:
These animals are fluorescent in all gut cells as result of an
inserted transgene CYP35A3 promotor::GFP. This is an integrated line.
3. Mount (as for Nomarski, Module 6) and examine strains by epifluorescence using
higher magnifications (20x and 40x).
17
Module 8 Gene inactivation by RNAi (bacterial feeding)
Timmons and Fire (1998) (see also Timmons et al., 2001) demonstrated
that it is possible to elicit RNAi effects by feeding worms bacteria expressing genespecific dsRNAs. In this module we will explore the effects of feeding worms bacteria
expressing dsRNAs corresponding to the C. elegans sex determining genes fem-1and
tra-2. In addition RNAi effects of unc-22 were tested. (Detailed methods for growth and
propagation of feeding constructs can be obtained at the Fire lab web site:
http://www.ciwemb.edu).
unc-22
The unc-22 gene encodes a serine-threonine protein kinase that may
regulate contraction. The enzyme is involved in myosin regulation may be
involved in regulating final stages of sarcomere assembly. Mutants are
uncoordinated and exhibit uncontrolled twitching of body-wall muscle
cells, muscle cells have disordered myofilament lattices.
fem-1
The fem-1 mutant is a sex-determination mutant, defective in spermatogenesis. Consequently, XX animals grown at the restrictive temperature
mature into females rather than hermaphrodites: they fail to make sperm.
As a result, they are self-sterile, and will produce no progeny by
themselves, but they can be fertilized by males. This is one way of
ensuring out-crossing, as opposed to selfing.
tra-2
The tra-2 gene normally promotes XX hermaphrodite development; lossof-function mutations in the tra-2 gene transform XX hermaphrodites into
non-mating males, but does not affect XO male development.
P450 31A2 This cytochrome P450 form is expressed in the embryogenesis, loss-offunction mutation in the gene results in a embryonic lethal phenotype.
The function of the encoded protein and the regulatory pathway are still
unknown.
Day 1
1. Inoculate overnight cultures of the different strains HT115(DE3) + plasmid in
LB+antibiotics (75 µg/ml ampicillin for amp-resistant plasmids and 12.5 µg/ml
tetracycline for selection of the HT115 strain). Incubate at 37°C with shaking
overnight.
18
Day 2
2. Dilute culture 1:50 in LB + antibiotics and grow to OD600 = 0.4. (A 10 ml culture is
usually enough for a small experiment). Induce by adding sterile IPTG (1 M stock
soöution) to 0.4 mM. Incubate 37°C with shaking for 4 hours.
3. Harvest cells by centrifugation (10 min, 4000 rpm), discard 5 ml of the supernatant
and resuspend the bacterial pellet in the remaining 5 ml. Spike the suspension with
additional antibiotics (another 75 µg/ml ampicillin and 12.5 µg/ml tetracycline) and
IPTG (to final total concentration of 0.8 mM)
4. Seed small agar plates with 70 µl of the prepared culture, let air dry the plates
under sterile box.
5. Place 4 L1/2 hermaphrodites on NGM-FEM, NGM-TRA, NGM-UNC and NGMP450 that have been seeded with bacteria expressing fem-1, tra-2 unc-22,
P45035C1 dsRNAs, respectively. Place in addition 4 L1/2 hermaphrodites on a
control plate. Put plates in 25°C and incubate over the weekend.
Day 4 and onward.
6. Examine parental worms and progeny for the following phenotypes:
NGM-UNC:
NGM-FEM:
NGM-TRA:
NGM-P450
Control:
Hermaphrodites show an UNC (uncoordinated) phenotype.
Hermaphrodites that have been transformed into females.
Hermaphrodites that have been partly or completely masculinized.
Hermaphrodites show wild type phenotype, but all eggs are dead
Wild type
19
Module 9
Extraction of RNA from worms
In this module, total RNA is prepared from a frozen pellet of worms, using
TRIZOL extraction. TRIZOL reagent is a solution of guanidine isothiocyanate and
phenol which simplifies the original method published by Chomczynski and Sacchi
(1987).
Day 2
1. Take packed frozen worms in a 15 ml polypropylene Falcon, add 1 Vol. RNasefree glass beads and taw the sample at 37°C water bath.
2. Vortex 6 x for 30 s, with occasional inversion of the tube to solubilize and lyse the
worms. Freeze (liquid nitrogen) 7 taw the worm after each votex step. Leave at RT
for 5 min.
3. Add 2 Vol. chloroform / ml worm pellet. Invert/vortex for 15 sec.
4. Spin 4,000 rpm for 10 min to remove insoluble material and separate phases.
5. Transfer supernatant to a fresh tube, add the same Vol. of chloroform.
Invert/vortex for 15 sec.
6. Spin 4,000 rpm for 10 min.
7. Transfer upper aqueous phase to a sterile centrifugation tube. Add 0.7 Vol.
isopropanol, invert, incubate at least 1 h at –20°C to precipitate RNA.
8. Spin 20,000 rpm for 20 min at 4°C.
9. Carefully remove supernatant. (Pellet will be very white).
10. Wash pellet with 15 ml of 75% ethanol. Vortex briefly. NOTE: Pellet will often float
free. Also RNA pellets can be stored in the 75% ethanol at –80°C for up to one
year safely.
11. Spin at 20,000 rpm for 5 min.
12. Remove supernatant and air dry pellets for 5-10 min.
13. Dissolve pellets in 50 µl RNase-free -H2O and freeze in the -70°C deep-freezer.
Day 9
Now the RNA has to be purified using an RNeasy plus mini kit from Qiagen including a
DNase step. Please note: Add 1/100 Vol. β-Mercaptoethanol to the RLT buffer before
use. Work under the hood!
14. Taw the RNA sample on ice, add 300 µl RLT buffer (β-Me added).
20
15. Transfer the sample to a gDNA Eliminator spin column placed in a 2 ml
collection tube (supplied in the kit). Incubate for 5 min. Centrifuge for 30 s at
10,000 rpm. Reload the flowthrough and repeat this step 1x.
16. Discard the column, and save the flowthrough. Make sure that no liquid
remains on the column membrane after centrifugation. If necessary, repeat
the centrifugation until all liquid has passed through the membrane.
17. Add 1 volume (350 µl) of 70% ethanol to the flowthrough, and mix well by
pipetting. Do not centrifuge.
18. Transfer up to 700 µl of the sample, including any precipitate that may have
formed, to an RNeasy spin column placed in a 2 ml collection tube (supplied
in the kit). Close the lid gently, and centrifuge for15 s at 10,000 rpm. Discard
the flow-through.
19. Add 700 µl Buffer RW1 to the RNeasy spin column. Close the lid gently, and
centrifuge for 15 s at 10,000 rpm to wash the spin column membrane. Discard the
flow-through.
20. Add 500 µl Buffer RPE to the RNeasy spin column. Close the lid gently, and
centrifuge for 15 s at ≥8000 x g (≥10,000 rpm) to wash the spin column
membrane. Discard the flow-through.
21. Repeat step 20
22. Centrifuge the empty tube including the column at full speed for 1 min.
(Perform this step to eliminate any possible carryover of Buffer RPE, or if
residual flow-through remains on the outside of the RNeasy spin column)
23. Place the RNeasy spin column in a new 1.5 ml collection tube (supplied).
Add 30 µl RNase-free water directly to the spin column membrane. Close
the lid gently, and centrifuge for 1 min at 10,000 rpm to elute the RNA.
24. Repeat the last step using the eluate from step 23; reuse the collection tube.
25. Freeze the sample in the -70°C deep-freezer.
21
Module 10 Temperature-shifts on temperature-sensitive mutants
C. elegans can grow productively at any temperature between 12°C and
25.6°C. 15°C and 25°C are used as the standard high and low temperatures.
Temperature-sensitive mutations provide a useful means of propagating mutations in
essential genes, and can also be used to generate populations consisting entirely of
inviable or sterile animals, by means of a temperature-shift. This module demonstrates
this, using two commonly-used ts-mutants, glp-4(bn2) and fem-1(hc17).
The glp-4 mutant is specifically defective in germline proliferation.
Consequently, animals grown at the restrictive temperature (25°C) develop into adults
with normal somatic development, but no germ cells. They are completely sterile.
The fem-1 mutant is a sex-determination mutant, defective in
spermatogenesis. Consequently, XX animals grown at the restrictive temperature
mature into females rather than hermaphrodites: they fail to make sperm. As a result,
they are self-sterile, and will produce no progeny by themselves, but they can be
fertilized by males. This is one way of ensuring out-crossing, as opposed to selfing.
The fer-1 mutant is fertilization defective. Sperm are produced but are
non-functional. Phenotypically mutant XX fer-1 animals resemble fem-1 mutants, but
the presence of sperm causes oogenesis to continue constitutively. Consequently
unfertilized oocytes are laid in large numbers. Laid oocytes are brownish and nonrefractile, unlike fertilized eggs. DAPI staining would reveal the presence of
endomitotic oocytes in the uterus. Strains such as this can be used for producing
preparations of pure oocytes in biochemical quantities.
Day 8
1. You will be provided with plates of WT, glp-4(ts), fem-1(ts) and fer-1(ts) which have
been maintained at 15°C. From each plate, pick 3 L4 hermaphrodites to a single
small spread NGM plate. Incubate at 25°C.
Day 9
2. Check that eggs are being laid by the hermaphrodites
Day 10
3. Examine the adult population. For both mutants, observe that there are many
adults, but these are all sterile or infertile, in contrast to the control WT population.
In the glp-4 mutant, the absence of a developed germline can be seen with a good
dissecting microscope. In the fem-1 mutant, stacks of unferilized oocytes
accumulate in the gonad, and can give a recognizable “stripy” appearance to the
ventral side of the animal. On the plates of fer-1 mutants, unfertilized oocytes can
be seen in large numbers
4. Add 5 - 10 wild type males to each of the two mutant plates, leave at 25°C.
Day 11
5. Examine the two plates. There should still be no eggs on the glp-4(ts) plate, but
many eggs on the fem-1(ts) and fer-1(ts) plate.
22
Module 11 lacZ staining of reporter transgene strains
lacZ staining is used to reveal expression patterns from reporter genes
carrying lacZ fusions. In this module, a strain carrying lacZ fused to a heat-shock
promoter (PK118) is induced before staining. Also, two strains carrying assorted lacZ
reporters are provided.
Day 10
1. Heat-shock strain PK118 for 45 min in 33°C waterbath, preferably late in the day.
Day 11
2. Heat shock strain PK118 again; wait a few hours
3. Stain PK118, UL6 and UL8 strains for beta-galactosidase (see below).
UL strains:
UL6
excretory cell, and nuclei of the hypodermis that lie close to the excretory
cell branches
UL8
spermathecae, 3 rectal epithelial cells
Staining worms (all stages)
•
Wash worms off plate with 2 ml water, place in 2 ml microfuge 30 sec 2000 rpm,
remove most of supernatant. Add 2 ml water and repeat spin. Carefully remove as
much as possible of supernatant with a pipetman. Freeze tubes on dry ice. When
all teams have placed their tubes on dry ice, they will be lyophilized (45 min).
[Alternatively, worms can be transferred to a glass slide in a minimal volume of
water, frozen on dry ice, and desiccated.]
•
Add a drop of cold acetone, allow evaporating. Add 200 µl staining solution,
incubate at 37°C. Periodically take a small volume of worm suspension and
examine by dissecting scope to monitor the progress of the staining (check about
timing). Some constructs may require 24 hr to develop blue color.
[Alternatively, if using the slide method, slides containing worms will be incubated
with staining solution in a humid chamber at 37°C.]
23
Module 12 Ballistic transformation of C .elegans
Vorbereitung der Würmer zum Schießen:
2-3 Wochen vor dem eigentlichen Schussexperiment werden kleine (35
mm) verhungerte Platten für das Animpfen großer (90mm) Platten vorbereiten um
möglichst viele gleichaltrige Würmer zu bekommen: Dafür je 5 L4 Würmer auf eine
kleine beimpfte Platte setzen und verhungern lassen (viele L1 Würmer). Die großen
Platten werden mit 1/4 bzw. 1/6 einer kleinen verhungerten Platte angeimpft. Pro Schuss
benötigt man etwa 1 große gut bewachsene Platte mit jungen, adulten Tieren (sie
sollten 3-10 Eier enthalten).
Für den Schuss werden kleine Platten benötigt, die in der Mitte einen
kreisrunden 25 μl großen und nicht zu stark angewachsenen OP50-Tropfen besitzen,
(auf denen werden später die Würmer zum Beschießen pipettiert). Es wird eine kleine
Platte pro Schuss benötigt.
Day 1
DNA-Gold Vorbereitung:
•
1 mg Goldpartikel in ein 1,5 ml Eppendorfgefäß einwiegen.
•
100 μl einer 50 mM Spermidinlösung dazugeben, vortexen und für 5-10 sec in ein
Ultraschallbad geben (volle Intensität).
•
anschließend 16 μg DNA dazu pipettieren, 10 min inkubieren und dabei öfter das
Eppendorfgefäß mit dem Finger “aufschnippen“.
•
danach wir das DNA-Goldgemisch auf 360 μl mit A. dest. aufgefüllt, gevortext und
für weitere 10 min inkubiert, dabei wieder einige Male das Eppendorfgefäß mit
dem Finger „aufschnippen“.
•
nach der Inkubation 100 μl 1M CaCl2-Lösung tropfenweise dazugeben (es darf
nicht klumpen) und für 10 Minuten präzipitieren.
•
anschließend 15-30 sec bei 13000 rpm abzentrifugieren und den Überstand mit
Hilfe einer Pipette entfernen. Den restlichen Überstand (ca. 10 μl) vorsichtig
aufmischen.
•
zum Schluss 3x mit 1 ml 96% Ethanol waschen und in 200 μl PVP-Lösung
aufnehmen.
• pro Schuss werden 20 μl eingesetzt (1 Ansatz reicht für 7-8 Schüsse)
Transformation:
•
Die kleinen beimpften Platten vom Vortag auf Eis stellen.
24
•
Pro Schuss benötigt man etwa 1 große (90mm) gut bewachsene Platte mit jungen
Adulten. Sind die Würmer im richtigen Alter, sie sollten 3-10 Eier enthalten, werden
sie mit 4-5 ml M9 Puffer von den Platten gespült und in ein 50 ml Falconröhrchen
überführt, wo man sie bei RT sedimentieren lässt. Der Überstand wird
abgenommen und verworfen.
•
Für das Schießen werden die Würmer vom Boden des Pellets mit einer
abgeschnittenen Eppendorfspitze abgenommen, in ein großes Eppendorfgefäß
überführt und mit M9 Puffer 2:1 verdünnt. Mit der gleichen Spitze (hoch- und
runterziehen!) werden 20 μl dieser Verdünnung auf den OP50-Tropfen der
vorgekühlten Platten pipettiert. Die Platten werden anschließend für weitere 2-3
Minuten auf Eis inkubiert, so dass das Wurmpellet fest wird.
•
in 80 % Alkohol eingelegte Düsen + Filterplättchen (pro DNA Probe ein Set) 15
min vor dem Schießen herausnehmen und trocknen lassen, kurz vor dem Schuss
zusammenbauen und 2-3x mal leer schießen.
Parameter:
Heliumdruck:
Pulszeit:
Vakuum:
Abstand Platte-Düse:
8 bar
10-30 ms
< 0,5-0,6 bar
≈12 cm
•
Düse wieder abbauen und auseinander schrauben, 20 µl (durchmischte) DNAGold Probe auf das Plättchen pipettieren, Düse wieder anbauen.
•
vorgekühlte und vorbereitete Platte ohne Deckel zentrisch auflegen, Kanone
schließen, Vakuum anlegen und anschließend den Schuss auslösen.
•
Platte entnehmen, mit Deckel schließen und bei 15°C inkubieren.
•
Nach Abschluss aller Transformationen die Agarschicht jeder beschossenen Platte
in acht gleiche Teile zerschneiden und einzeln auf große mit Bakterien versehene
Platte überführen, bei 15°C inkubieren
Day 3
•
Platten in den 25°C Brutschrank stellen.
Day 8 and onward
•
Suche nach deutlich kleinen Würmern (L1-L3), einzeln auf eine kleine Platte
abpicken und jeweils bei 25°C inkubieren.
Referenz:
Wilm et al., 1999
25
Module 13 Single worm PCR
Für die Auswertung des Moduls 14 (Ballistic transformation) erfolgt der
Nachweis über das Vorhandensein der GFP-DNA in den potentiell transgenen Tieren
mittel Single worm PCR. Mit dieser Methode ist es möglich, den transgenen Status der
Nematoden auch ohne eine direkten Nachweis der GFP Produktion nachzuweisen.
Dies ist insbesondere bei solchen Varianten von entscheidender Bedeutung, von
denen es noch unbekannt ist, ob überhaupt ein GFP Produktion möglich ist.
Protokoll:
Day 11
In Abhängigkeit der Anzahl von pha-1 positiven Nematoden (lebende F1 bei der
restriktiven Temperatur von 25°C) werden möglichst viele der nun adulten Tiere wider
von der Selektionsplatte abgepickt.
1. Einen adulten Wurm von der Platte abpicken und in ein mit 2 µl Single worm lysis
Mix versehenes mittleres PCR-Eppi überführen.
(Überprüfung mit dem Mikroskop)
2. Eppis für 30 min bei – 80°C einfrieren
3. jeweils Zugabe von 1-2 Tropfen Mineralöl und Proben für 1 h bei 60°C und
anschließend 15 min bei 95°C erwärmen (im Thermozykler)
4. Proben auf Eis stellen, Zugabe von 23 µl PCR-Mix je Tube in die untere Phase, nur
diese wässrige Phase anschließend 2x hoch und runter pipettieren.
1,8 µl
1,5 µl
0,5 µl
0,25 µl
0,25 µl
0,12 µl
18,58 µl
----------23,00 µl
10x PCR Puffer (QIAGEN)
MgCl2 (25 mM)
dNTP-Mix (10 mM)
sense Primer GFP
antisense Primer GFP
HotStarTaq-Polymerase (QIAGEN)
A. dest (steril)
5. PCR nach folgendem Regime ablaufen lassen:
95°C
5 min
1 cycle
95°C
56°C
72°C
45 sec
45 sec
45 sec
35 cycles
72°C
10 min
1 cycle
6. Gelauswertung: 10-20 µl der unteren wässrigen Phase zusammen mit 1/6 Vol.
Probenverdünnungspuffer auf ein Agarosegel auftragen und auswerten.
• Agarose in der Mikrowelle erwärmen und vollständig schmelzen
(Achtung: Vorsicht bei Siedeverzug !)
• Elektrophorese-Apparatur zusammensetzen, Agarose auf rund 60°C abkühlen
lassen
26
ETHIDIUMBROMID IST KANZEROGEN; Arbeiten mit Nitril-Handschuhen !
• Ethidiumbromid Stammlösung zum Agarose-Gel hinzufügen (3,75 µl / 50 ml);
Gel in die Apparatur hineingießen
• Agarose-Gel 30 min bei Raumtemperatur abkühlen und fest werden lassen.
• Begrenzungsblöcke und Kämme vorsichtig entfernen, Gel samt Gelträger in
die Elektrophoresekammer einsetzen.
• Elektrophoresekammer soweit mit 1 x TAE Puffer füllen, bis das Gel gerade
bedeckt ist.
• Aufzutragende DNA-Probe im Verhältnis 1:6 mit Probenverdünnungspuffer
mischen und von oben gerade in den einzelnen Slot hineinpipettieren
• Elektrophoresekammer schließen und bei einer Spannung von rund 100 V für
15-40 min laufen lassen. (Polung: – nach +)
• Gel unter dem AlphaImager auswerten.
Puffer:
Wurm-Lysispuffer
10 mM Tris-HCl
50 mM KCl
2.5 mM MgCl2
0,45% NP40
0,45% Tween 20
0,01% Gelatine
Single worm lysis Mix (frisch ansetzen)
100 µl Wurm-Lysispuffer
1 µl Proteinase K Lösung (10mg/ml)
Agarose
0,7-2,0 % Agarose
für 400 ml:
gelöst in 1x TAE Puffer
8 ml
50 x TAE Puffer
2,0 M Tris / Acetat
50 mM EDTA
Ethidiumbromid
Stammlösung
10 mg Ethidiumbromid / ml Aqua dest.
6x Probenverdünnungspuffer
auf 1l
pH 8,3
3,4- 8,0 g Agarose
50 x TAE Puffer
242 g Tris
57.1 ml Essigsäure (konz., Eisessig)
100 ml 0,5 M EDTA (pH = 8,0)
(Endkonzentration 0,75 mg/ml Gel)
40 %
Sacharose
10 mM Bromphenolblau
27
Module 14 Reproduction and thermo-tolerance
Reproduktionstest
Einige Substanzen beeinflussen die Reproduktion von C. elegans hinsichtlich der
Nachkommenszahl und des Zeitpunkts der Haupt-Eiablage. Häufig wird die These
vertreten, dass Substanzen, die eine längere Lebensspanne hervorrufen zugleich eine
verminderte Reproduktion bewirken.
Um eine derartige Wirkung nachzuweisen, werden Würmer dem jeweiligen Stoff
ausgesetzt. Dies geschieht, indem man einerseits die zu untersuchende Konzentration
der Substanz beim Gießen in den Agar gibt und andererseits den Futterbakterien, die
auf die Platten gegeben werden, zusetzt (Compound-Platten mit Futterbakterien
werden jeweils bereitgestellt)
Die Nachkommen werden pro Tag und pro behandelten Tier gezählt und mit der Zahl
der F1 von Kontrolltieren (unbehandelt) verglichen.
Es werden folgende Substanzen getestet: Rosmarinsäure und Catechin jeweils in
drei verschiedenen Konzentrationen (100, 200, 300 µM). Jeder Student testet dabei
nach Absprache nur eine Konzentration eines Stoffes (10 Platten) und eine
Kontrollcharge (10 Platten).
Der Zeitpunkt, an dem die Würmer in der 1 .Generation auf die Platten mit der
Substanz gebracht werden, beeinflusst die Wirkung maßgeblich. Daher wird hier mit
der 2.Generation gearbeitet um so die Zeitkomponente abzuschwächen und den
Versuch zu vereinfachen. Bei dem Versuch hier wird mit L4 Larven begonnen.
Tag 1: 10 behandelte L4-Würmer auf je eine Compound-Platte setzen und 10
unbehandelte L4-Würmer auf je eine unbehandelte NGM-Platte setzen. Platten
beschriften und im Wärmeschrank bei 20 °C aufbewahren.
Tag 2: Jeden adulten Wurm auf eine neue Platte transferieren und sowohl die alten als
auch die neuen Platten bei 20 °C inkubieren.
Tag 3: Jeden adulten Wurm auf eine neue Platte transferieren und sowohl die alten als
auch die neuen Platten bei 20 °C inkubieren. Platten von Tag 1 kontrollieren (in
wieweit sind F1 entwickelt, welches Stadium?) und gegebenenfalls (ab L3/L4) abends
in den Kühlschrank stellen, damit sich die Entwicklung verzögert und die Platten am
nächsten Tag ausgezählt werden können.
Tag 4: Jeden adulten (parentalen) Wurm auf eine neue Platte transferieren und sowohl
die alten (Tag 3) als auch die neuen Platten (Tag 4) bei 20 °C inkubieren. Die
Nachkommen auf den ersten Platten (Tag 1) mit auszählen. Die Platten vom Tag 2
überprüfen, gegebenenfalls (s. o.) abends in den Kühlschrank stellen oder direkt
auszählen.
(Am Wochenende: Die Platten der Tage 3 und 4 in den Kühlschrank stellen, wird
veranlasst)
Tag 8: Die Nachkommen der Platten von den Tagen 2 bis 4 werden ausgezählt.
28
Thermotoleranz
Häufig ist verlängertes Leben assoziiert mit einer erhöhten Stresstoleranz. Zudem
kann sich der Hormesis-Effekt auch bei Fehlen einer lebensverlängernden Wirkung
durch eine höhere Toleranz gegenüber verschiedenen Stressoren bemerkbar machen.
Um die Stresstoleranz zu testen, werden die Tiere mehrere Stunden einer erhöhten,
lebensbedrohlichen Temperatur ausgesetzt und die Überlebenden in der behandelten
Charge mit denen der Kontrolle verglichen.
Es werden die gleichen Substanzen wie beim Reproduktionstest verwendet
(Rosmarinsäure und Catechin). Jeder Student testet wieder die gleiche Konzentration des Stoffes, die er auch beim Reproduktionstest hatte (2 Platten). Zusätzlich hat
jeder wieder eine Kontrollcharge (2 Platten).
Auch hier werden Würmer verwendet, die in zweiter Generation auf dem jeweiligen
Stoff wachsen. Dadurch kann ebenfalls mit L4 Larven begonnen werden.
Tag 4: 2 x 30 L4-Würmer von Compound-Platten auf 2 neue Compound-Platten
setzen und 2 x 30 Kontroll-Tiere auf 2 neue NGM-Platten setzen.
(Am Wochenende: tägliches Umsetzen der Tiere auf neue Platten)
Tag 8: Umsetzen der Tiere auf 4 neue Platten. Die Anzahl der Würmer pro Platte
notieren.
(In der Nacht von Tag 9 zu Tag 10: Alle Platten in den 35 °C Brutschrank stellen, wird
veranlasst)
Tag 10: Nach 10-stündigem thermalen Stress werden tote und lebende Würmer
ausgezählt. Jedes Tier, was sich auch nach Berührung mit dem Picker nicht bewegt,
wird als tot gewertet.
29
Module 15 Informatics resources
There will be a general introduction to Wormbase and some special
internet resources (individually at Day 9 or 10) with demonstration exercises.
C. elegans Databases
AceDB - A C. elegans Database (acedb.org)
ACeDB was written by Richard Durbin and Jean Thierry-Mieg as part of the C. elegans
genome mapping and sequencing project. This hugely successful genome database
program has replaced the paper versions of the genetic and physical maps in many C.
elegans labs. In addition, the AceDB database engine has become the standard for
other genomic mapping projects. In addition to genetic and physical map data AceDB
contains:
•
•
•
•
•
genomic sequence
gene predictions
worm literature
ESTs
in situ hybridisation data
AceDB can be installed on Windows but it runs best on the UNIX/Linux platform.
There are two official AceDB distribution FTP sites at which you can get the latest full
release of the software as source code or pre-compiled executables.
•
•
ftp://ftp.sanger.ac.uk/pub/acedb/
ftp://ncbi.nlm.nih.gov/repository/acedb/
Wormbase (www.wormbase.org)
WormBase is a repository of mapping, sequencing and phenotypic information about
the C. elegans nematode. This prototype of the final database is layered on top of
ACeDB, and has not been subjected to the rigorous curation expected of the ultimate
product. The data available correspond to the July 2001 release and contain the
"essentially complete" genomic sequence. This site is updated regularly.
Wormbase vs AceDB:
Advantages
•
•
•
Wormbase uses a web-browser such as Netscape/Internet Explorer.
There is no need install or update software/data.
Links to other relevant sites
Disadvantages
•
•
Need web access
Less flexible than AceDB for displaying data.
30
WWW Resources
C. elegans WWW Server (www.c.elegans.leeds.ac.uk or elegans.swmed.edu)
This site is the central C. elegans web resource. It is maintained by Leon Avery in the
US and mirrored in the UK by David Coates. It contains links to most C. elegans
resources as well as a worm literature search engine, a researcher list, collections of
recent worm papers, worm community announcements and general information about
C. elegans and the worm community.
The Caenorhabditis Genetic Center at the University of Minnesota
(biosci.umn.edu/CGC/CGChomepage.htm)
The Caenorhabditis Genetics Center (CGC) is supported by the National Institute of
Health National Center for Research Resources. The CGC has been in operation since
1978, first at the University of Missouri, Columbia and since 1992 at the University of
Minnesota, St. Paul. The main operations of the CGC are at the University of
Minnesota in St. Paul. Robert K. Herman is the director; Theresa Stiernagle is the
curator. The CGC also has two subcontractors: Jonathan Hodgkin in England, and
Leon Avery at the University of Texas Southwestern Medical Center in
Dallas, TX.
The St. Paul team is responsible for collecting, maintaining, and distributing stocks of
C. elegans, maintaining a C. elegans Bibliography, and publishing and distributing the
Worm Breeder's Gazette. The UK team is responsible for coordinating genetic
nomenclature and maintaining the C. elegans genetic map. The Dallas team is
responsible for maintaining the C. elegans web server.
This web-site has a search engine to search for strains of bacteria or nematodes
available from the CGC as well as nomenclature guidelines, a C. elegans bibliography
and the Worm Breeder’s Gazette Archives. The CGC maintains several thousand
stocks representing most of the standard mutants and genomic rearrangements, which
are made available free to academic researchers.
The C. elegans EST database (www.ddbj.nig.ac.jp/c-elegans/html/CE_INDEX.html)
This database contains cDNA information generated by Yuji Kohara’s lab in Japan. It
also includes the “The Nematode Expression Pattern DataBase”. The Kohara lab has
been constructing an expression pattern map of the 100Mb genome of the nematode
Caenorhabditis elegans through EST analysis and systematic whole mount in situ
hybridization. NEXTDB is the database to integrate all information from our expression
pattern project. Information available in the current version is as follows;
•
•
•
•
Map: Visual expression of the relationships among the cosmids, predicted
genes and the cDNA clones.
Image: In situ hybridization images arranged by developmental stages.
Sequence: Tag sequences of the cDNA clones are available.
Homology: Results of BLASTX search are available.
31
Jim Kent's (Zahler Lab) Intronerator (www.cse.ucsc.edu/~kent/intronerator)
A collection of tools for exploring the molecular biology and genomics of C. elegans
with a special emphasis on alternative splicing. The Intronerator also provides
alternate gene predictions in addition to aligning cDNAs with gene predictions.
WormPD TM by Proteome (www.proteome.com/databases/WormPD)
A commercial web-site that collates biological information about worm proteins.
Information can be queried by protein name, sequence or data category. WormPD
contains :
•
•
•
•
•
Total Proteins 19675
Total References 2724
Proteins Characterized by Genetics or Biochemistry 1936
Proteins Known by Homology to Characterized Proteins 9346
Proteins of Unknown Function 8393
RNAi Experiments
The results of most RNAi experiments can be found in Wormbase or AceDB.
Microarray Data (cmgm.stanford.edu/~kimlab/wmdirectorybig.html)
Microarray data and techniques can be found at Stewart Kim’s web-page. It links to the
Stanford microarray database.
The bionet.celegans newsgroup
To subscribe, see http://www.elegans.swmed.edu/
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References
Reference Books
Wood, W.B. et al. (eds.) (1988). The nematode C. elegans. Cold Spring Harbor
Laboratory Press
Riddle, D.L. et al. (eds.) (1997). C. elegans II. Cold Spring Harbor Laboratory Press
Epstein, H.F., and Shakes, D.C. (eds.) (1995). Caenorhabditis elegans: Modern
Biological Analysis of an Organism. Methods in Cell Biology Vol. 48, Academic Press
Hope, I. (ed.) (1999) Caenorhabditis elegans: A Practical Approach Oxford University
Press
Specific References
Brenner S (1974) The genetics of Caenorhabditis elegans. Genetics 77:71-94
Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid
guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156-159
Jakubowski J, Kornfeld K (1999) A local high-density, single-nucleotide
polymorphism map used to clone Caenorhabditis elegans cdf-1. Genetics 153: 743752.
Timmons L, Fire A (1998) Specific interference by ingested dsRNA. Nature 395: 854.
Timmons L, Court DL, Fire A (2001) Ingestion of bacterially expressed dsRNAs can
produce specific and potent genetic interference in Caenorhabditis elegans. Gene 263:
103-112.
Wicks SR, Yeh RT, Gish WR, Waterston RH, Plasterk RH (2001) Rapid gene
mapping in Caenorhabditis elegans using a high density polymorphism map. Nat
Genet 28: 160-164.
Williams BD, Schrank B, Huynh C, Shownkeen R, Waterston RH (1992) A genetic
mapping system in Caenorhabditis elegans based on polymorphic sequence-tagged
sites. Genetics 131: 609-624
Wilm T, Demel P, Koop HU, Schnabel H, Schnabel R (1999) Ballistic transformation
of Caenorhabditis elegans. Gene 229: 31-35.
Manual Reference
This manual is mainly based on protocols from the 31st Wellcome Trust Advanced
Course “Genetic, Molecular and Informatic, Methods for C. elegans”.
http://www.wellcome.ac.uk/en/1/bioseradv31.html