transgenic animals - technology and applications
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TRANSGENIC ANIMALS - TECHNOLOGY AND APPLICATIONS
GOETZ LAIBLE, AGRESEARCHBIOLOGY TEACHERSPROFESSIONAL DEVELOPMENT DAY28TH MARCH 2013
PRESENTATION OUTLINE
International TG livestock examples
Historical perspective
TG livestock projects at AgResearch Ruakura
Main Technologies
LOOKING BACK
1980 Transgenic mice by MI 1985 Transgenic sheep, pigs and rabbits by MI 1989 Gene KO in mice
2000 Gene KO in sheep
1977 Human insulin produced in bacteria
1996 Cloning of Dolly the sheep by SCNT
1973 Genetic engineering in bacteria
2003 GloFish, first transgenic animal approved for commercialisation
2006 First animal-produced human drug approved
1974 Transgenic mice, virus-mediated
1997 Transgenic sheep by SCNT
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
3. 2.
Transfection of bovine cells
Generation of cloned transgenic cattle
2.
Transfection of bovine cells
Oocyte enucleation
Generation of cloned transgenic cattleGeneration of cloned transgenic cattle
3. 2.
Transfection of bovine cells
Oocyte enucleation
Fusion of donor cell and cytoplast
Generation of cloned transgenic cattleGeneration of cloned transgenic cattle
3. 2.
Transfection of bovine cells
Oocyte enucleation
Fusion of donor cell and cytoplast
Activation of reconstructed
embryo
Generation of cloned transgenic cattleGeneration of cloned transgenic cattle
3. 2.
Transfection of bovine cells
Oocyte enucleation
Fusion of donor cell and cytoplast
Embryo culture to blastocyst
Activation of reconstructed
embryo
Generation of cloned transgenic cattleGeneration of cloned transgenic cattle
3. 2.
Transfection of bovine cells
Oocyte enucleation
Fusion of donor cell and cytoplast
Embryo culture to blastocyst
Activation of reconstructed
embryo
Generation of cloned transgenic cattle
Embryo transfer
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 TALENsIntroduction of specific double strand breaks
T TTTTT TTTTTT
TTTTTTTTTTTTTTTTTTTTTTTT
small deletions small insertions
Non homologous end joining
MOLECULAR BREEDING
Elite genotypes, deleting genes, replacing genes, ……
Homologous recombination
T TTTTT TTTTTT
T TTTTT TTTTTT
T TTTTT TTTTTT
genetically modified ESCs
Artificial Gametes
In vitro fertilization
In vitro differentiation
POSSIBILITIES WITH EMBRYONIC STEM CELLS (ESCs)
Breeding
Embryo aggregation
Chimeric mouse
.
Nuclear Transfer Embryos
Embryo transfer
Nuclear transfer
Transgenic mouse
Available only for mouse and rat
TG LIVESTOCK - A PLATFORM TECHNOLOGY FOR A VARIETY OF APPLICATIONS
Biomedical 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 Increased production Sustainability of intensive farming Disease resistance Production of novel foods
Gene of InterestInstructions1) DNA construct
Secretion into milkActivated in the lactating mammary gland
4) Extraction of the pharmaceutical protein from milk and use as a drug for
disease treatments
2) Stable integration of the DNA construct into the genome
3) Mammary gland produces large amounts of
proteins that are readily accessible in milk
THE CONCEPT OF BIOPHARMING
BIOPHARMING – ALREADY A REALITY
ATryn®
GTC Biotherapeutics2006 EMA2009 FDA
RuconestTM
Pharming 2010 EMA
PIGS FOR XENOTRANSPLANTATIONDisruption 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 improvementsMulti-gene transgenics to control complement-mediated lysis and inflammation, and coagulation
Dai et al., 2002
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
Cystic fibrosisCFTR KO and common human mutation ∆508Recapitulate devastating lung infections
EXAMPLES OF LIVESTOCK DISEASE MODELS
Huntington’s diseaseHTT transgene with 73 Q repeat
Rogers et al., Science 2008Pezzulo ey al., Nature 2012
Jacobsen et al., Hum Mol Genet 2010
NON-BIOMEDICAL APPLICATIONS HAVE LOWER ACCEPTANCE
AGRICULTURAL/FOOD APPLICATIONSProduction GH salmonαLac pigs
Animal WelfareBSE, FMD, mastitis resistant cattle
Sustainable farmingEnviro pig
Food with health benefitsOmega-3 pigshLF, hLZ, cattle, goats
Devlin et al., Nature 1994Nobel et al., J Anim Sci 2002
Richt et al., Nat Biotechnol 2007Wang et al., Plos One 2012Wall et al., Nat Biotechnol 2005
Golovan et al., Nat Biotechnol 2001
Lai et al., Nat Biotechnol 2006Van Berkel et al., Nat Biotechnol 2002Maga et al., J Dairy Sci 2006
Chris Slane, Farmers Weekly, 15 October 2012
GM LIVESTOCK RESEARCH IN NEW ZEALAND
NUCLEAR TRANSFER TECHNOLOGY
HIGHLY VOCAL OPPONENTS OF GE TECHNOLOGY
STRICT REGULATORY REQUIREMENTSGM 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 CommitteeRegular 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
mRNA (working copy)TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
TCTGTT
T
G
T
C
T
A
TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTT
T
TA DNA (information)
MECHANISM OF RNA INTERFERENCE (RNAi)
Proteins (the worker)
Nucleus
Cell
micro RNA
micro RNA geneInstructions1) DNA construct
Activated in the lactating mammary gland
4) The mammary gland produces milk that no longer contains BLG
2) Stable integration of the DNA construct into the genome
3) The micro RNA blocks the production of BLG in
the mammary gland
THE CONCEPT OF REDUCING BLG IN MILK
BLG KNOCK DOWN IN DAIRY CATTLE
Daisy (with micro RNA)
BLG
Conve
ntion
al milk
(w
/o micr
o RNA)
Daisy m
ilk
(w
ith m
icro R
NA)
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 tailChen 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
Daisy with no tail
Nuclear Transfer
Daisy’s twin foetus
.
.
Daisy’s cells Daisy’s twins’ cells
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