Transcript Slide 1

TRANSGENIC ANIMALS - TECHNOLOGY AND
APPLICATIONS
GOETZ LAIBLE, AGRESEARCH
BIOLOGY TEACHERS
PROFESSIONAL DEVELOPMENT DAY
28TH MARCH 2013
PRESENTATION OUTLINE

Historical perspective

Main Technologies

International TG livestock examples

TG livestock projects at AgResearch Ruakura
LOOKING BACK
 1973 Genetic engineering in bacteria
 1974 Transgenic mice, virus-mediated
 1977 Human insulin produced in bacteria
 1980 Transgenic mice by MI
 1985 Transgenic sheep, pigs and rabbits by MI
 1989 Gene KO in mice
 1996 Cloning of Dolly the sheep by SCNT
 1997 Transgenic sheep by SCNT
 2000 Gene KO in sheep
 2003 GloFish, first transgenic animal approved
for commercialisation
 2006 First animal-produced human drug approved
 2009 Gene KO rats with designer nucleases
 2011 Gene KO pigs with designer nucleases
TECHNOLOGICAL ADVANCES IN LIVESTOCK
TRANSGENESIS
Pronuclear Microinjection
Somatic cell nuclear transfer
(SCNT) with transfected cells
2.
3.
Generation of cloned transgenic cattle
Transfection of
bovine cells
Generation
cloned transgenic
cattlecattle
Generation
of ofcloned
transgenic
Transfection of
bovine cells
2.
Oocyte
enucleation
Generation
cloned transgenic
cattlecattle
Generation
of ofcloned
transgenic
Transfection of
bovine cells
2.
Oocyte
enucleation
3.
Fusion of donor
cell and cytoplast
Generation
cloned transgenic
cattlecattle
Generation
of ofcloned
transgenic
Transfection of
bovine cells
Activation of
reconstructed
embryo
2.
Oocyte
enucleation
3.
Fusion of donor
cell and cytoplast
Generation
cloned transgenic
cattlecattle
Generation
of ofcloned
transgenic
Embryo culture
to blastocyst
Transfection of
bovine cells
Activation of
reconstructed
embryo
2.
Oocyte
enucleation
3.
Fusion of donor
cell and cytoplast
Generation of cloned transgenic cattle
Embryo transfer
Embryo culture
to blastocyst
Transfection of
bovine cells
Activation of
reconstructed
embryo
2.
Oocyte
enucleation
3.
Fusion of donor
cell and cytoplast
TECHNOLOGICAL ADVANCES IN LIVESTOCK
TRANSGENESIS
Pronuclear Microinjection
Somatic cell nuclear transfer
(SCNT) with transfected cells
Zinc finger nucleases (ZFNs)
Transcription activator-like
effector nucleases (TALENs)
TECHNOLOGICAL ADVANCES IN LIVESTOCK
TRANSGENESIS
Pronuclear Microinjection
Somatic cell nuclear transfer
(SCNT) with transfected cells
Zinc finger nucleases (ZFNs)
Transcription activator-like
effector nucleases (TALENs)
LATEST TOOLS – CUSTOM NUCLEASES
ZFNs and TALENs
Introduction of specific double strand breaks
Non homologous end joining
TTTTTTTTTTTT
TTTTTTTTTTTT
small deletions
small insertions
MOLECULAR BREEDING
TTTTT
T
T
TTTTT
TTTTTT
TTTTTT
Homologous recombination
TTTTTT
TTTTTT
Elite genotypes, deleting genes, replacing genes, ……
POSSIBILITIES WITH EMBRYONIC STEM CELLS (ESCs)
Available only for mouse and rat
genetically modified ESCs
Nuclear transfer
Nuclear Transfer Embryos
Embryo transfer
Embryo aggregation
Chimeric mouse
Breeding
Transgenic mouse
In vitro differentiation
Artificial Gametes
In vitro fertilization
TG LIVESTOCK - A PLATFORM TECHNOLOGY
FOR A VARIETY OF APPLICATIONS
Biomedical
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Biopharming
Medical/functional foods
Xenotransplantation
Animal models of human
diseases
Agricultural
 Improved quantity and
quality of animal production
 Improved animal health
 Sustainable agriculture
RELEVANT INTERNATIONAL EXAMPLES
Biomedical applications
 Biopharming
 Xenotransplantation
 Human disease models
Agricultural applications
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Increased production
Sustainability of intensive farming
Disease resistance
Production of novel foods
THE CONCEPT OF BIOPHARMING
1) DNA construct
Instructions
Gene of Interest
Activated in the lactating mammary gland
Secretion into milk
2) Stable integration of the DNA construct into the genome
3) Mammary gland produces
large amounts of proteins
that are readily accessible
in milk
4) Extraction of the pharmaceutical protein
from milk and use as a drug for disease
treatments
BIOPHARMING – ALREADY A REALITY
ATryn®
GTC Biotherapeutics
2006 EMA
2009 FDA
RuconestTM
Pharming
2010 EMA
PIGS FOR XENOTRANSPLANTATION
Disruption of the gene (α1,3GT) that is
responsible for marking pig cells as
foreign and causing the hyperacute
rejection of transplant organs
First of a series of downstream hurdles
that need to be overcome for extending
the survival of organ transplants
De-cellularized medical device
applications (surgical mesh, heart valves)
are strong product candidates
Forms basis for further improvements
Dai et al., 2002
Multi-gene transgenics to control
complement-mediated lysis and
inflammation, and coagulation
LARGE ANIMAL MODELS FOR HUMAN DISEASES
Mice have been the model of choice but
differences in size and physiology to
humans can be major shortcomings
Size, life span, physiology of large
animals are more similar to humans
Enables study of chronic degenerative
disease processes and testing of new
therapeutic strategies and drugs
EXAMPLES OF LIVESTOCK DISEASE MODELS
Cystic fibrosis
CFTR KO and common human mutation ∆508
Recapitulate devastating lung infections
Rogers et al., Science 2008
Pezzulo ey al., Nature 2012
Huntington’s disease
HTT transgene with 73 Q repeat
Jacobsen et al., Hum Mol Genet 2010
NON-BIOMEDICAL APPLICATIONS HAVE LOWER
ACCEPTANCE
AGRICULTURAL/FOOD APPLICATIONS
Production
GH salmon
αLac pigs
Devlin et al., Nature 1994
Nobel et al., J Anim Sci 2002
Animal Welfare
BSE, FMD, mastitis resistant cattle
Richt et al., Nat Biotechnol 2007
Wang et al., Plos One 2012
Wall et al., Nat Biotechnol 2005
Sustainable farming
Enviro pig
Golovan et al., Nat Biotechnol 2001
Food with health benefits
Omega-3 pigs
hLF, hLZ, cattle, goats
Lai et al., Nat Biotechnol 2006
Van Berkel et al., Nat Biotechnol 2002
Maga et al., J Dairy Sci 2006
GM LIVESTOCK RESEARCH IN NEW ZEALAND
Chris Slane, Farmers Weekly, 15 October 2012
NUCLEAR TRANSFER TECHNOLOGY
HIGHLY VOCAL OPPONENTS OF GE TECHNOLOGY
STRICT REGULATORY REQUIREMENTS
GM Animals are regulated under the Hazardous Substances and New Organisms
(HSNO) Act by the Environmental Protection Authority (EPA)
Long term (until 2030) regulatory approval to develop GM animals in outdoor
containment
Sheep
Goats
Cattle
Facilities and activities audited by the Ministry of primary Industries (MPI)
All research involving animal requires the approval by an Animal Ethics Committee
Regular engagement with Māori liaison group
THE DAISY PROJECT
 2-3 % of infants are allergic to cows’ milk proteins
 The whey protein beta-lactoglobulin (BLG) is thought to be the main
allergen in cows’ milk
 BLG is not produced in humans (or mice) and can elicit a strong immune
response
 Transgenic technology offers the potential to eliminate or reduce allergy
causing proteins like BLG
 Testing the feasibility of harnessing the natural mechanism of RNA
interference to reduce the amount of BLG in cows’ milk
RNA INTERFERENCE
Highly conserved cellular mechanism thought to have evolved
as a defence mechanism against endogenous parasitic or
invading pathogenic DNA/RNA
Triggered by the presence of short double-stranded RNAs
Target recognition involves sequence-specific recognition of
selected messenger RNAs
A specific class of interfering RNAs (micro RNAs) have
emerged as important regulators of the expression of
endogenous genes
MECHANISM OF RNA INTERFERENCE (RNAi)
A CG T
DNA (information)
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
T GC A
Nucleus
Cell
mRNA (working copy)
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
micro RNA
Proteins (the worker)
THE CONCEPT OF REDUCING BLG IN MILK
1) DNA construct
Instructions
micro RNA gene
Activated in the lactating mammary gland
2) Stable integration of the DNA construct into the genome
3) The micro RNA blocks the
production of BLG in the
mammary gland
4) The mammary gland produces milk that
no longer contains BLG
BLG KNOCK DOWN IN DAIRY CATTLE
BLG
Daisy (with micro RNA)
Visualised milk proteins
BUT MISSING A TAIL
 Taillessness is a known rare congenital defect
in cattle
 One report of a cloned calf born without a tail
Chen et al., Cell Res 2011
Is it caused by the:
- transgene insertion
- transgene expression
- transgene-independent mutation
- cloning (epigentic reprogramming) error?
RECLONING DEMONSTRATES EPIGENTIC CAUSE
miRNA 6-4 bovine cell line
Nuclear Transfer
Daisy’s twins’ cells
Daisy’s cells
Daisy’s twin
foetus
Daisy with
no tail