Transgenic Animals - Technology and Applications
GOETZ LAIBLE, AGRESEARCH MWC - BIOLOGY TEACHERS PROFESSIONAL DEVELOPMENT DAYS 6/7TH APRIL 2017
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)
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Scientific/Technical Staff 405 FTE
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Science Groups • Animal Science (cattle, goats, sheep, deer) • Food & Bio-based Products • Forage Science • Farm Systems & Environment • Knowledge & Analytics
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
TECHNOLOGICAL ADVANCES IN LIVESTOCK TRANSGENESIS Pronuclear Microinjection (1985)
CLONING BY NUCLEAR TRANSFER
Dolly the sheep, 1996
Wells et al., Biology of Reproduction, 1999
TECHNOLOGICAL ADVANCES IN LIVESTOCK TRANSGENESIS Pronuclear Microinjection
Somatic cell nuclear transfer (SCNT) with transfected cells (1997)
2.
3.
GENERATION OF CLONED TRANSGENIC CATTLE Promoter
Transfection of bovine cells
Transgene
GENERATION OF CLONED TRANSGENIC CATTLE
Transfection of bovine cells
2. Oocyte enucleation
GENERATION OF CLONED TRANSGENIC CATTLE
Transfection of bovine cells
2. Oocyte enucleation
3. Fusion of donor cell and cytoplast
GENERATION OF CLONED TRANSGENIC CATTLE
Transfection of bovine cells
Activation of reconstructed embryo
2. Oocyte enucleation
3. Fusion of donor cell and cytoplast
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
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
THE CONCEPT OF BIOPHARMING 1) DNA construct
Promoter
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® (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
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
SCNT
Puro
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
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
ELIMINATING AN ALLERGY CAUSING PROTEIN PROTEIN
BLG Daisy (with micro RNA)
Visualised milk proteins
Jabed et al., PNAS, 2012
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)
THE CRISPR REVOLUTION Scientific publications mentioning CRISPR, 2007-2017
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* As of March 2017
GENOME EDITING WITHOUT FOOTPRINT ZFNs, TALENs and CRISPRs Introduction of specific double strand breaks
Non homologous end joining (NHEJ)
TTTTTTTTTTTT TTTTTTTTTTTT
small deletions
small insertions
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
WT
KO Whitworth et al., Nature Biotechnology, 2016
MOLECULAR BREEDING TTTTT T
T
TTTTT TTTTTT TTTTTT
Homologous recombination
TTTTTT TTTTTT Rapid generation of elite genotypes by directly introducing beneficial natural mutations
ACCESSING THE BEST VARIANTS OF ALL GENES
Heat tolerant
Hornless
HORNLESS DAIRY CATTLE
Horned dairy cattle Polled beef cattle
Polled dairy cattle
Tan et al., PNAS, 2013 Carlson et al., Nature Biotechnology 2015
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
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Cell-mediated editing (correction) with ZFNs
Shanthalingam et al., PNAS 2016
TUBERCULOSIS-RESISTANT CATTLE Enhancing transgenesis with genome editing tools
Wu et al., PNAS 2015
ANIMALS FOR TRANSPLANT ORGANS
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
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
Chris Slane, Farmers Weekly, 15 October 2012
