1. Lec 13 & 14 Animal biotechnology
Shah Rukh Abbas, PhD

2. Transgenic animals
Transgenics are genetically modified organisms with DNA from another source inserted in to their genome
A large number of transgenic animals have been created mice, cows, pigs, sheep goats fish frogs and insects etc.
Currently no transgenic animal or animal product is approved by the FDA for human consumption.

3. Why you GM animals? Research purposes Improve live stock animals
Use of animals as bioreactors

4. I. Animals in research

5. I. Animals in research FDA oversights of ‘drug development process’
Animals involved in pre-clinical research and development

6. A. Cloning

7. Cloning: I. Embryo Twinning

8. Cloning: II. Somatic Cell nuclear transfer

9. Dolly

10. Limitations of cloning
Decrease genetic diversity
Epigenetic effects
Abnormal development
Not much efficient and costly
Premature aging

11. Future of cloning Increase in genetic gain Conserve endangered species
Consistent quality

12. B. Creating Transgenic animals

13. Production of Transgenic animals Methadology
Step 1 – Construction of a transgene
Transgene made of 3 parts:
Pomoter
Gene to be expressed
Termination sequence
Step 2 – Introduction of foreign gene into the animal
Pronuclear microinjection method
Embryonic stem cell method.

14. Methods of transferring genes
Via
Microinjection
Recombinant defective retrovirus
Linker based sperm mediated gene transfer
Transgenic animals are then produced/cloned using following techniques
Embryo stem cell method
Pronuclear injection
SCMT

15. I.

16. II. Embryonic Stem cell method

17. a.

18. b.

19. Step 3: Screening for transgenic positives
Transgenic progenies are screened by PCR to examine the site of incorporation of the gene
Some transgenes may not be expressed if integrated into a transcriptionally inactive site.
Step 4: Further animal breeding is done to obtain maximal expression.
Heterozygous offsprings are mated to form homozygous strains.

20. Mass production via SCNT

21. Limitations of transgenic animal generation
Some of the drawbacks of there methods are:
The inserted DNA randomly integrates in to genome which may lead to silencing of an essential gene or its lethality
Insertion into a different area can affect the gene regulation
The eggs must be harvested and fertilised in vitro
More than one copy of the gene may get in to the genome.

22. Examples of transgenic animals I. Transgenic cattle (for biopharming)

23. II. Transgenic fish Superfish Glo fish Increased growth and size
Growth hormone gene inserted into fertilized egg.
Transgenic salmon grows about 10 – 11 times faster than normal fish.
Glo fish
GM freshwater zebra fish (Danio rerio)
Produce by integrating a fluorescent protein gene from jelly fish into embryo of fish.

25. Anti Freeze Proteins (AFPs)

26. III. Transgenic mouse In the brain of Alzheimer’s patients, dead
Alzheimer’s mouse
In the brain of Alzheimer’s patients, dead
nerve cells are entangled in a protein
called amyloid.
Mouse made by introducing amyloid
precursor gene into fertilized egg of mice.
Oncomouse
Mouse model to study cancer
Made by inserting activated oncogenes.
Smart mouse
Biological model engineered to overexpress NR2B receptor in the synaptic pathway.
This makes the mice learn faster like juveniles throughout their lives.

27. IV. TRANSGENIC PIG V. Transgenic rabbit
Enviro pig
Pigs have trouble fully digesting a
compound known as phytate found in many cereal grains used to feed them.
Transgenic pigs are created by introducing phytase gene
of E.coli.
Phytase enzyme is thus produced in the salvary gland of pig
It degrades indigestible phytate with the release of phosphate that is readily digested by pigs.
Pig for organ transplant
Pigs with human genes, in order to decrease the chance of organ rejection by human body.
V. Transgenic rabbit
Alba, the EGFP (Enhanced Green Flurescent protein) bunny
Created in 2000 as a transgenic artwork.

28. VI. Transgenic cattle For good quality wool production.
Bioreactors whose cells have been engineered to synthesis marketable proteins.
More economical than producing desired protein in cell culture.
Transgenic cattle
Transgenic cows are made to produce proteins lactoferrin and interferons in their milk.
Prion free cows resistant to mad cow disease.
Transgenic sheep
For good quality wool production.
Transgenic goat
Goats that could express tissue plasminogen
activator, anti thrombin III, spider silk etc in milk.

29. Biopharming
Transgenic animals for production of human or veterinary pharmaceuticals.
Expression of the transgene in the mammary gland of large farm animals like cow, goats and sheep offers advantages like easy isolation of the product secreted into the milk and low cost-large scale production.
therapeutic proteins against osteoporosis, cystic fibrosis, hemophilia, malaria, arthritis, HIV etc. 

31. Issues related to Transgenic animals
Blurring the lines between species by creating transgenic combinations.
There may be health risks associated with transgenics.
There may be long term effects on the environment when transgenic animals are released into the field.
Various bioethicist argue that it is wrong to create animals that would suffer as a result of genetic alteration.

32. Recent trends
Transgenic goats engineered to produce human breast milk ( Journal Transgenic Research, August 2012)
University of California scientists created the transgenic goats by transferring human genes for breast milk enzymes and proteins into goat embryos.
Produce 60 percent of the lysozyme and lactoferrin found in human mother’s milk.
For babies of mothers who aren’t present, or can’t nurse them, milk from these transgenic goats could provide the next-best alternative.
Bioluminescent transgenic mouse model for study mammary gland tumour development. (PLoS ONE, aug 2012)
Research done by Cancer Biology and Therapeutics Group,  Ireland