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Transgenic Animals - Technology and applications

Transgenic Animals - Technology and applications. Goetz Laible, AgResearch Biology Teachers Professional Development day 28 th March 2013. Presentation outline. Historical perspective. Main Technologies. International TG livestock examples.

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Transgenic Animals - Technology and applications

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  1. Transgenic Animals - Technology and applications • Goetz Laible, AgResearch • Biology Teachers • Professional Development day • 28th March 2013

  2. Presentation outline • Historical perspective • Main Technologies • International TG livestock examples • TG livestock projects at AgResearch Ruakura

  3. 1973 Genetic engineering in bacteria • 1974 Transgenic mice, virus-mediated Looking Back • 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

  4. technological advances in livestock transgenesis Pronuclear Microinjection 2. 3. Somatic cell nuclear transfer (SCNT) with transfected cells

  5. Generation of cloned transgenic cattle Transfection of bovine cells

  6. Generation of cloned transgenic cattle Generation of cloned transgenic cattle Transfection of bovine cells 2. Oocyte enucleation

  7. Generation of cloned transgenic cattle Generation of cloned transgenic cattle Transfection of bovine cells 2. 3. Oocyte enucleation Fusion of donor cell and cytoplast

  8. Generation of cloned transgenic cattle Generation of cloned transgenic cattle Transfection of bovine cells Activation of reconstructed embryo 2. 3. Oocyte enucleation Fusion of donor cell and cytoplast

  9. Generation of cloned transgenic cattle Generation of cloned transgenic cattle Embryo culture to blastocyst Transfection of bovine cells Activation of reconstructed embryo 2. 3. Oocyte enucleation Fusion of donor cell and cytoplast

  10. Generation of cloned transgenic cattle Embryo transfer Embryo culture to blastocyst Transfection of bovine cells Activation of reconstructed embryo 2. 3. Oocyte enucleation Fusion of donor cell and cytoplast

  11. 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)

  12. 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)

  13. Latest Tools – custom nucleases ZFNsand TALENs Introduction of specific double strand breaks T T T T T T T T T T T T Non homologous end joining T T T T T T T T T T T T T T T T T T T T T T T T small deletions small insertions

  14. Molecular Breeding T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T Homologous recombination T T T T T T Elite genotypes, deleting genes, replacing genes, ……

  15. Possibilities with Embryonic stem cells (ESCs) Available only for mouse and rat genetically modified ESCs Embryo aggregation Nuclear transfer In vitro differentiation . Nuclear Transfer Embryos Chimeric mouse Artificial Gametes Embryo transfer Breeding In vitro fertilization Transgenic mouse

  16. 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

  17. relevant international examples • Biomedical applications • Biopharming • Xenotransplantation • Human disease models • Agricultural applications • Increased production • Sustainability of intensive farming • Disease resistance • Production of novel foods

  18. 1) DNA construct Instructions Gene of Interest The Concept of Biopharming 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

  19. Biopharming – already a reality ATryn® GTC Biotherapeutics 2006 EMA 2009 FDA RuconestTM Pharming 2010 EMA

  20. 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 Multi-gene transgenics to control complement-mediated lysis and inflammation, and coagulation Pigs for xenotransplantation Dai et al., 2002

  21. 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 Large Animal Models for human Diseases

  22. Examples of livestock Disease Models Cystic fibrosis CFTR KO and common human mutation ∆508 Recapitulate devastating lung infections Rogers et al., Science 2008 Pezzuloey al., Nature 2012 Huntington’s disease HTT transgene with 73 Q repeat Jacobsen et al., Hum Mol Genet 2010

  23. Non-biomedical applications have lower acceptance

  24. Production GH salmon αLac pigs Animal Welfare BSE, FMD, mastitis resistant cattle Sustainable farming Enviro pig Food with health benefits Omega-3 pigs hLF, hLZ, cattle, goats Agricultural/Food applications Devlin et al., Nature 1994 Nobel et al., J AnimSci 2002 Richt et al., Nat Biotechnol 2007 Wang et al., Plos One 2012 Wall et al., Nat Biotechnol 2005 Golovan et al., Nat Biotechnol 2001 Lai et al., Nat Biotechnol 2006 Van Berkel et al., Nat Biotechnol 2002 Maga et al., J Dairy Sci 2006

  25. GM livestock research in new zealand Chris Slane, Farmers Weekly, 15 October 2012

  26. Nuclear transfer technology

  27. highly vocal opponents of GE technology

  28. 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

  29. 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

  30. 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

  31. T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T A C G T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T C A G DNA (information) Mechanism of RNA interference (RNAi) Nucleus Cell mRNA (working copy) micro RNA Proteins (the worker)

  32. 1) DNA construct Instructions micro RNA gene The Concept of Reducing BLG in milk 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

  33. BLG KNOCK DOWN IN DAIRY CATTLE Conventional milk (w/o micro RNA) Daisy milk (with micro RNA) BLG Daisy (with micro RNA) Visualised milk proteins

  34. 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?

  35. 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

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