Transgenic Animals - Technology and applications - Home | …€¦ · PPT file · Web view · 2015-09-10Generation of cloned transgenic cattle. Generation of cloned transgenic cattle.

TRANSGENIC ANIMALS - TECHNOLOGY AND APPLICATIONS

GOETZ LAIBLE, AGRESEARCHBIOLOGY TEACHERSPROFESSIONAL DEVELOPMENT DAY28TH MARCH 2013

http://gateway/resources/corpaffairs/lowland/Farm/cows.jpg

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.


Oocyte enucleation

Generation of cloned transgenic cattleGeneration of cloned transgenic cattle

3. 2.


Oocyte enucleation

Fusion of donor cell and cytoplast


3. 2.


Oocyte enucleation


Activation of reconstructed

embryo


3. 2.


Oocyte enucleation


Embryo culture to blastocyst


embryo


3. 2.


Oocyte enucleation


Embryo culture to blastocyst


embryo

Generation of cloned transgenic cattle

Embryo transfer




Zinc finger nucleases (ZFNs)

Transcription activator-like effector nucleases (TALENs)




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

Transgenic Animals - Technology and applications - Home | …€¦ · PPT file · Web view · 2015-09-10Generation of cloned transgenic cattle. Generation of cloned transgenic cattle.

Documents