Bioreactors and transgenic animals

Bioreactors and transgenic animals
Ryszard Slomski, Daniel Lipinski, Marlena Szalata,
Joanna Zeyland, Jacek Jura, Zdzislaw Smorag
Poznan University of Life Sciences, Poznan, Poland
Institute of Human Genetics, PAS, Poznan, Poland
National Research Institute of Animal Production, Cracow, Poland
TRENDS IN SCIENCE AND TECHNOLOGY
RELEVANT TO THE BIOLOGICAL AND TOXIN WEAPONS
CONVENTION
31 October─3 November 2010
Institute of Biophysics, Chinese Academy of Sciences
Beijing, China
Bioreactors
Production methods
Bacteria
Yeast
Fungi
Plants
Cell
cultures
Animals
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Total costs
Post-translational
modifications
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Up-scaling
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Regulations
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Speed
Bioreactors
Specific aims
1. Selection of valuable protein for production.
2. Selection of targeted site of transgenesis.
3. Selection of organism for transgenesis
Yield of production per year/required volume
Processing potentials
Culturing/breeding problems
Utilization of the recombinant product
Time needed to manufacture product
Bioreactors
From idea to product
Screening of bacterial cultures
overexpressing gene construct
1 2 3
4 5 6 7 8 9 10 11 12 13 14 15
Purification under native (lanes 1-8) or denaturing conditions (lanes 915). Lanes 1,2,3,9,10,11,12, different clones with FeldI chain 1 protein,
lanes 4,5,6,12,13,14, different clones with FeldI chain 2, lanes 7,8,15,
controls. Lanes 12,13,14 expression 106 amino acid peptide in E.coli.
Purification of FeldI proteins overexpressed in
E.coli by metal affinity chromatography
FeldI chain 1
1 2 3 4 5 6
FeldI chain 2
7 8
1 2 3 4
5
6
7 8
Left site: overexpression of FeldI chain 1 protein. Lane 1, control; lane
2, overexpression; lane 3, proteins not bound to column; lanes 4-7,
fractions of purified protein; lane 8, marker (16.9, 14.4, 8.2 Da).
Right site: overexpression of FeldI chain 2 protein. Lane 1, control;
lane 2, overexpression; lane 3, proteins not bound to column; lanes 47, fractions of purified protein; lane 8, marker (16.9, 14.4, 8.2 Da).
Overexpression and purification of
recombinant Glu and Cat domain of S. mutans
1
2
3
4
5
6
7
8
9
10
Purification of recombinant Glu by
metal affinity chromatography. Lane 1,
cell lysate; lane 2, unbound protein;
lanes 3-9, elutions; lane 10, weight
marker (14.4-116 kDa). Recombinant
Glu peptide is indicated by an arrow.
1
2
3
4
5
6
7
8
9
Purification of recombinant Cat by metal
affinity chromatography. Lane 1, cell
lysate; lane 2, unbound protein; lanes 38, elutions; lane 9, weight marker (14.4116 kDa). Recombinant Cat peptide is
indicated by an arrow.
Animal bioreactors
Animal systems for production
 Blood
 Urine
 Seminal plasma
 Egg white
 Silk worm cocoon
 Milk
Animal bioreactors
Exemplary categories of polypeptides
 Growth factors
 Transmembrane regulators
 Hormones
 Immunoglobulins
 Antiviral proteins
 Milk lipases
 Lipocortins
 Cell surface proteins
 Lipotropins
 Human pancreatic enzymes
 Interleukins
 Enkephalins
 Interferons
 Silk proteins
 Stimulating factors
 Spider silk proteins
 Kinases
Animal bioreactors
Silkworm larvae
Silkworm (Bombyx mori) - natural silk manufacturer for
textile industry
Cocoon – potential
recombinant proteins
source
of
high
amounts
of
Short time of generation
Production of vaccines - cholera toxin B subunit fusion
protein linked with human insulin B chain peptide at
levels up to 0.97 g/l of hemolymph)
Animal bioreactors
Expression analysis
Expression of LacZ gene of
pBRJZ vector in embryonic
tissues of Bombyx mori
subjected to transgenesis by
biollistic method.
A.
B.
C.
D.
E.
F.
G.
H.
Embryo
Brain
Brain section
Brain section
Silk producing gland
Silk producing gland section
Gonad
Gastrointestinal tract and
body cover
J. Thomas, 2003
Animal bioreactors
Mammary gland
Value addition (increase one of the casein
components in milk for production of cheese or
yogurt)
Milk of higher nutrient content (alteration of milk
composition has the potential to enhance the
production of certain proteins and/or growth factors
that are deficient in milk)
Production of proteins affecting human and animal
health (human butyrylcholinesterase, mastitis)
Animal bioreactors
Mammary gland
Rabbit (human IGF-1, human tissue plasminogen
activator, erythropoietin, α-glucosidase, factor NGF-β,
protein C, human growth hormone, rotavirus inner core
proteins, human factor VIII, human alpha 1,3
fucosyltransferase)
Goat (human lactoferrin, E2–CSFV vaccine, human tissue
plasminogen activator, human antithrombin III, human
monoclonal antibodies, growth hormone)
Sheep (human factor VIII, human factor IX, human α-1antitrypsin, fibrinogen)
Pigs (protein C, human factor VIII)
Cows (human lactoferrin, human erythropoietin, human
serum albumin)
Animal bioreactors
Designing of gene construct
 Promoter and functional gene
 Detection system
 Purification system
Animal bioreactors
WAP:FUC rabbits
Animal bioreactors
Comparison of the production of milk using different
transgenic animal species
Animal
Pregnancy
(months)
Maturation
(months)
Months from
Amount of milk
per lactation (l) microinjection to
milk production
Mouse
0.75
1
0.0015
3-6
Rabbit
1
5-6
1-1.5
7-8
Pig
4
7-8
200-400
15-16
Sheep
5
6-8
200-400
16-18
Goat
5
6-8
600-800
16-18
Cow
9
15
8000
30-33
Animal bioreactors
High efficiency of expression
Proper posttranslational modifications
Low maintaining costs
High reproductivity of transgenic founders
Breeding in pathogen free environment
Lack of human infecting agents
Animal bioreactors
Detection & stability
Transgenic rabbits 05 and 08
Generation F1
Animal bioreactors
Final stage of transgenesis - production
Homozygous transgenic female 12B
Transgenesis was confirmed by molecular and cytogenetic analysis
Animal bioreactors
Recombinant protein purification
1
2
3
4
5
6
7
8
43 kDa
30 kDa
17.2 kDa
12.3 kDa
Purification of human growth hormone from milk of transgenic
rabbit. Samples of milk collected from lactating females were
subjected to metal affinity column chromatography (Talon).
Animal bioreactors
Blastomere cloning
Animal bioreactors
ATryn® - recombinant human antithrombin
ATIII, goat milk, GTC Biotherapeutics
ATryn®, recombinant form of human antithrombin, is the
first transgenically produced protein to be approved
anywhere in the world, having recently been approved
by the European Commission for the prophylactic
treatment of deep vein thrombosis in patients with
hereditary
antithrombin
deficiencies
that
are
undergoing surgical procedures.
Xenotransplantation
Waiting list
Comparison of number of patients waiting for transplants of
vascularized organs with number of transplantations. The U.S. Organ
Procurement and Transplantation Network and the Scientific Registry
of Transplant Recipients, Annual Report 2008
Xenotransplantation
Cell surface antigens
Structure of carbohydrate ends of AB0 antigens and
α1,3Gal [Gal(α1,3)Gal] epitope
Xenotransplantation
Epitope Gal evolution
CH
OH
2
5
1
O
1,3
l
Ga
3
3
Gal
4
6
O
4
6CH 2OH
5
2
2
1
A
Men
Cattle
Pig
B
Great Apes
Old World Monkeys
New World Monkeys
hGT
Men
Cattle
Pig
Mouse
ABO
Great Apes
Old World Monkeys
hGT
New World Monkeys
Mouse
ABO
Phylogenetic
evolution
tree
of
epitope
α1,3Gal.
Human
galactosyltransferase gene (HGT2) originated before differentiation of
New World Monkeys (A) or after differentiation (B)
D. Lipinski, R. Slomski
Xenotransplantation
Specific aims
1. Gene constructs for inactivation of specific genes.
2. Gene constructs for modification of specific genes.
3. Gene constructs for regulation of specific genes.
Xenotransplantation
Cloning of genes
1. Preparation of transcript.
2. Synthesis of cDNA.
3. Cloning in vectors.
28 S
FUT 1098 bp
GAL 1290 bp
18 S
5S
Initial RNA preparations
Amplification of human α1,2-fucosyltransferase
(FUT) gene and human α-galactosidase (GAL) gene
Xenotransplantation
Preparation of gene construct
Vector
Insert
Gene construct encoding human α1,2fucosylotransferase
under
CMV
promoter, competing with endogenous
α1,3-galactosyltransferase for the same
substrate N-acetyllactosamine.
α1,2-FUT
Restriction analysis of pCMVFUT gene
construct. Vector was hydrolyzed with
XbaI and BamHI enzymes (cloning sites of
modified
sequence
encoding
α1,2fucosyltransferase in pGT-N29 vector,
containing cloned CMV promoter and
poly(A) of hGH gene).
Xenotransplantation
Microinjection
Xenotransplantation
Transgenic pigs
 Competitive gene constructs
α1,2 fucosyltransferase
α galactosidase
 Inactivating gene constructs
Disrupted α1,3 galactosyltransferase
 Regulatory gene constructs
Inhibitors of complement
TG1154
Xenotransplantation
CMVFUT transgene detection
pCMVFUT gene construct and products of two PCR reactions (144 bp
and 343 bp). Lanes 1-16, DNA of potentially transgenic pigs; lane 17,
negative control (-DNA); lane 18 (W), positive control (CMV:Fut gene
construct); lane 19 (M), size marker 267 bp and 745 bp.
Xenotransplantation
Mapping of CMVFUT transgene in F2 generation
F0
F2
F2
FISH localization of transgene on metaphase chromosomes of
transgenic pigs. On the left metaphase plate of heterozygote TG1154
boar pCMVFUT transgene on chromosome 14q28. On the right
metaphase plate of homozygote 433 pig, after TG1154 boar with
pCMVFUT transgene.
Stability of transgenesis.
F1 offspring
1154
(TG1154)
162
163
164
165
166
167
168
169
170
Negative control
(human DNA
Negative control
(no DNA)
Positive control
(pCMVFut)
Marker 5050-500
Xenotransplantation
Cytogenetics of transgenic animals
Nucleus of WAPhGH transgenic rabbit fibroblast was analyzed using 3D
FISH methods with confocal microscopy LSM 510 (analysis of xy axis).
Xenotransplantation
Expression of FUT transgene
1 2 3 4 5 6 7 8 9 10 11 12
RT-PCR analysis of expression of FUT transgene under control of CMV promoter in
ear specimens of TG1154 boar in comparison with control pigs (1152, 1155). 400 bp
long fragment of cDNA of α1,2-fucosyltransferase was amplified. Transgene was
indicated by arrow. Lanes 1-3, RNA purity control: lane 1, boar TG1154; lane 2, non
transgenic pig 1152; lane 3, non transgenic pig 1155; lanes 4-6, cDNA positive
controls, cDNA of β-actin gene: lane 4, boar TG1154; tor 5, non transgenic pig 1152;
lane 6, non transgenic pig 1155; lanes 7-9, detection of presence of cDNA of FUT
transgene: lane 7, transgenic boar TG1154; lane 8; negative control, non transgenic
pig 1152; lane 9; negative control, non transgenic pig 1155; lane 10, negative control
(no cDNA); lane 11, positive control (pCMVFUT plasmid); lane 12, size marker, DNA of
phage  hydrolyzed with HindIII and EcoRI enzymes.
Xenotransplantation
1 2 3 4
5 6
7
8 9 10 11
Expression of FUT transgene
RT-PCR analysis of expression of FUT
transgene under control of CMV promoter in
tissues of transgenic pig 166 (heart, kidney,
liver, skeletal muscle and ovary) in
comparison with tissues of control non
transgenic pig 167. 400 bp transcript
fragment of α1,2FT was amplified. Lanes 110, RNA purity control, β-actin genomic
sequence, RNA isolated from tissues of
transgenic and non transgenic pigs; lanes
12-21, cDNA positive control, tissues of
transgenic and non transgenic pigs, cDNA
sequence of β-actin gene (300 bp); lanes 2327, transgenic pig 166; lane 28-32, negative
control, tissues of non transgenic pig; lane
33, negative control, no cDNA; lane 34,
positive control, pCMVFUT plasmid; lane
11,22,35, size marker, DNA of phage 
hydrolyzed with HindIII and EcoRI enzymes
12 13 14 15 16 17 18 19 20 21 22
23 24 25 26 2728 29 30 31 32 33 34 35
Xenotransplantation
Statistical analysis of survival of transgenic cells
H antigen
Gal epitope
Flow cytometry analysis of fibroblasts isolated from control, transgenic
boar TG1154 and homozygous pig 433 after staining with UEA-1 lectin
(left) (detects H antigen) and BS-IB4 lectin (right) (detects Gal epitope).
The x-axis shows the fluorescence intensity, and the y-axis shows the
relative cell count for unstained cells (black thin dashed-dot line),
control (red thin solid line), boar TG1154 (blue thick solid line) and pig
433 (purple thick dotted line). The analysis demonstrates that increased
expression of H antigen correlates with reduced expression of Gal
epitope on the cell surface of transgenic pigs (boar TG1154 and pig 433).
Xenotransplantation
Statistical analysis of survival of transgenic cells
100%
90%
V
i
a
b
i
l
i
t
y
80%
70%
60%
nontransg enic
50%
triple-transgenic
40%
30%
20%
10%
0%
0%
20%
30%
40%
50%
Human serum
Mean values of the viability (%) of the non transgenic lines and
four transgenic lines (pCD46, pCD55 pCD59 and triple-transgenic)
in the human complement-mediated cytolysis assay.
Xenotransplantation
Recently developed pig with α-galactosidase gene
Creation of artificial life
“I am creating artificial life”, declared in
October 2007 US C. Venter researcher
involved in the race to sequence the human
DNA. He has built a synthetic chromosome
and announced the creation of the first new
artificial life form on Earth.
Venter "a very important philosophical step in the history of our species.
We are going from reading our genetic code to the ability to write it. That
gives us the hypothetical ability to do things never contemplated before".
Team of 20 top scientists, led by the H. Smith has constructed a synthetic
chromosome based on the bacterium Mycoplasma genitalium, that is 381
genes long and contains 580,000 base pairs of genetic code. The
synthetically reconstructed chromosome has bee named Mycoplasma
laboratorium. It was then transferred into a living bacterial cell and in
effect become a new life form.
The new life form will depend for its ability to replicate itself and
metabolize on the molecular machinery of the cell into which it has been
injected, and in that sense it will not be a wholly synthetic life form.
Pat Mooney, director of a Canadian bioethics organization, ETC Group:
"Governments, and society in general, is way behind the ball. This is a
wake-up call - what does it mean to create new life forms in a test-tube?"
Craig Venter responded: “We are trying to create a new value system for
life. When dealing at this scale, you cannot expect everybody to be
happy."
Thank you for your attention