Lec 13 & 14 Animal biotechnology Shah Rukh Abbas, PhD 19.2.2015 23.2.2015

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.

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

I. Animals in research

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

A. Cloning

Cloning: I. Embryo Twinning

Cloning: II. Somatic Cell nuclear transfer

Dolly

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

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

B. Creating Transgenic animals

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.

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

I.

II. Embryonic Stem cell method

a.

b.

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.

Mass production via SCNT

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.

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

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.

Anti Freeze Proteins (AFPs)

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.

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.

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.

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. 

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.

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

http://www.dnatube.com/video/5571/What-is-Animal-Biotechnology