1. Transgenic Animals - Technology and Applications
Goetz Laible, AgResearch
MWC - Biology Teachers
Professional Development days
6/7th April 2017

2. AgResearch – in a nutshell
Crown Research Institute in support of NZ’s pastoral industries
Four major research centres
Ruakura
Grasslands
Lincoln
Invermay
Total Staff 786 (full-time equivalent)
Scientific/Technical Staff 405 FTE
Science Groups
Animal Science (cattle, goats, sheep, deer)
Food & Bio-based Products
Forage Science
Farm Systems & Environment
Knowledge & Analytics

3. GM of 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

4. technological advances in livestock transgenesis
Pronuclear Microinjection (1985)

5. Cloning by Nuclear Transfer
Dolly the sheep, 1996
Wells et al., Biology of Reproduction, 1999

6. technological advances in livestock transgenesis
Pronuclear Microinjection 3. 2.
Somatic cell nuclear transfer (SCNT) with transfected cells (1997)

7. Generation of cloned transgenic cattle
Transgene
Promoter
Transfection of bovine cells

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

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

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

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

12. 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

13. The Concept of Biopharming
Gene of Interest
Promoter
1) DNA construct
Secretion into milk
Activated in the lactating mammary gland
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

14. Biopharming – already a reality
ATryn® (antithrombin alpha)
Congenital antithrombin deficiency
rEVO Biologics
2006 EMA/2009 FDA
RuconestTM (C1 esterase inhibitor)
Hereditary angioedema 
Pharming NV
2010 EMA/2014 FDA
KanumaTM (sebelipase alfa)
Lysosomal acid lipase deficiency
Alexion Pharmaceuticals
2015 EMA/2015 FDA

15. Goats Producing an anti-cancer mAB
Biosimilar antibody Cetuximab produced in the milk of goats
Erbitux approved for the treatment of metastatic colorectal cancer and head and neck cancer LC HC
Puro
SCNT
First generation antibodies are coming off patent
Antibody drugs are required in large amounts (dosage, patients)
Transgenic animal production systems are ideally suited for large scale production
Chimeric mouse/human monoclonal antibody that blocks human epidermal growth factor receptor
Innovator drug, currently produced in cultured murine cells
Approved for the treatment of metastatic colorectal cancer and head and neck cancer
Treatment costs are around NZD 50,000 per patient
Annual production of 200 kg (2005) and world wide market of $1.9 billion (2011)
Herd of 100 goats at our current production rates
= cetuximab
2 lines of goats (single female founders)

16. TG food animals – a different story
Salmon with enhanced growth characteristics
due to a growth hormone transgene
22 years in the regulatory maze
1989 – TG salmon generated
1993 – discussions with FDA initiated
1995 – application for approval filed
2009 – last supporting dataset submitted
2010 – classified as safe to eat and safe for the environment and recommended for approval
– process stalled again and no final decision announced for
another 5 years

17. 1st TG food animals approved
AquAdvantage salmon –approved for human consumption in November 2015
Blocked by pending federal bill in March 2016 requesting new impact study on wild salmon stocks

18. Eliminating an allergy causing protein protein
Conventional milk
(w/o micro RNA)
(with micro RNA)
Daisy milk
BLG
Daisy (with micro RNA)
Visualised milk proteins
Jabed et al., PNAS, 2012

19. Genetic engineering advances in livestock
Pronuclear Microinjection (1985)
Somatic cell nuclear transfer (SCNT) with transfected cells (1997)
Genome Editing
ZFNs Zinc finger nucleases
TALENs Transcription activator-like effector nucleases
CRISPRs Clustered regularly interspaced short palindromic repeat (CRISPR) – Cas9 nucleases (2012)

20. The crispr revolution
Scientific publications mentioning CRISPR, *
* As of March 2017

21. Genome editing without footprint
ZFNs, TALENs and CRISPRs
Introduction of specific double strand breaks T
Non homologous end joining (NHEJ) T
small deletions
small insertions

22. PRRSV-resistant pigs
(Procine Reproduction and Respiratory Syndrome Virus)
CRISPR-mediated disruption (1506 bp deletion) of the CD163 gene
Whitworth et al., Biology of Reproduction, 2014
porcine reproductive and respiratory syndrome virus WT KO
Whitworth et al., Nature Biotechnology, 2016

23. Molecular Breeding T T T T Homologous recombination
Rapid generation of elite genotypes by directly introducing beneficial natural mutations

24. Accessing the best variants of all genes
Hornless
Heat tolerant

25. Hornless Dairy Cattle Horned dairy cattle Polled beef cattle
Polled dairy cattle
Tan et al., PNAS, 2013
Carlson et al., Nature Biotechnology 2015

26. Shipping fever-resistant cattle
Introducing a ‘novel’ mutation
A one amino acid change in ruminants prevents signal peptide cleavage of CD18 and generates binding site for bacterial leukotoxin
Cell-mediated editing (correction) with ZFNs
Shanthalingam et al., PNAS 2016

27. Tuberculosis-resistant cattle
Enhancing transgenesis with genome editing tools
Wu et al., PNAS 2015

28. Animals for transplant Organs

29. 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

30. livestock Models of human Diseases
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

31. Chris Slane, Farmers Weekly, 15 October 2012

33. What are animal clones? genetically identical organisms
Cloning = producing clones, either naturally or artificially

34. Natural cloning: Twins

35. Cloning by embryo splitting
splitting embryos at the blastocyst (cattle) or 6–8 cell (human) stage

36. Reproductive medicine to cure metabolic mitochondrial diseases: The power of three
“Three person IVF”

37. Genome transplantation
Pronuclear Transfer
Maternal Spindle Transfer