1. PRODUCTION OF THERAPEUTICS FROM TRANSGENIC ANIMALS

2. CONTENTS:- Reasons for producing transgenic animals.
Historical background.
What is a transgenic animal.
Method for production of transgenic animals.
Study of transgene expression.
Applications.
References.

3. Reasons for producing transgenic animals :-
Obtain fundamental information on living organisms and human diseases.
Use animals as source of pharmaceutical proteins.
Improve animal production.
Animals are useful as disease models and producers of substances for human welfare.

4. Historical background :-
1970 first transgenic mice via, viral infection, but not germline transmission.
1980 first transgenic mice via, microinjection, the most popular technique.
1981 Gordon & Ruddle coined the term transgenic, animal containing foreign gene.
1985 first transgenic rabbits, sheep, pigs, & cattle.

5. What is a transgenic animal :-
A transgenic animal is one that carries a foreign gene that has been deliberately inserted into its genome . Foreign gene is constructed by recombinant DNA technology .
Along with a structural gene, DNA usually include other sequences like scorable & selectable marker genes.

6. Methods for production of transgenic animals :-
DNA microinjection.
Embryonic stem cell trasfer.
Targeted gene transfer.
Virus mediated gene transfer.

7. DNA microinjection
Direct microinjection of a chosen gene construct into pronuclear of a fertilized ovum.
first methods that proved to be effective in mammals.
Insertion of DNA is a random process, high probability that the introduced gene will not insert itself into a site on the host DNA that will permit its expression.
Manipulated fertilized ovum is transferred into the oviduct of a foster mother.
A major advantage of this method is its applicability to a wide variety of species.

9. Embryonic stem cell –mediated gene transfer
Make your DNA
Using recombinant DNA methods, built mol. of DNA having str. gene we desire
Vector DNA
Promoter & enhancer sequences.
Transform ES cells in culture
Select for successfully transformed cells.
Inject these cells into inner cell mass of mouse blastocysts.
Embryo transfer : prepare a pseudopregnat mouse & transfer embryos into her uterus.
Test her offspring :examine its DNA for desired gene
Establish a transgenic strain.
Chimeric mouse produced by ES Cell method

11. Targeted gene insertion
In this method transgene integrates at the precise site where its allele is present in the genome. this is achieved by homologous recombination.

12. Retrovirus-mediated gene transfer
. Earliest method for successful gene transfer in mammals.
Virus has transfection property.
Killed virus is replication defective
Virus gene is replicated with transgene gene
Can be used to transfect a wide range of cells e.g. ES cells, embryo cell
Method was successfully used in 1974 when a simian virus was inserted into mice embryos, resulting in mice carrying this DNA .

13. Detection of transgenic animals & transgene expression
Identification of transgenic animals:-It utilizes either dot- blot technique or PCR amplification using genomic DNAs extracted from tail biopsies from 6-7 week old mice. The-ive +ive selection by PCR given in diagram.

14. Analysis of transgene integration
The integration of transgene into the genome is confirmed by southern hybridization of genomic DNA extracted from transgenic individuals, DNA digested with a suitable restriction enzyme prior to electrophoresis. Choosing appropriate restriction enzyme for DNA digestion, the integration of transgene as well as no. of copies per cell & multiple integration site also established.

15. Detection of mRNA expression
. A high purity RNA preparation is obtain from the transgenic individuals and subjected to northern hybridization, which provides additional information on transcript size as well.

16. Transgenic animals applications
Transgenic mice –As disease model system :--- mice model for human genetic diseases such as Alzheimer’s disease, arthritis, muscular disorder, hypertension, & many other have been developed.
Trangenic mice that carry & express a transgene encoding betaA4 portion of APP which might provide a model for studying molecular basis of Alzheimer’s disease.
Transgenic mice used as model for expression system e.g. (CFTR) protein are needed to study its function & for treating cystic fibrosis, so transgenic mice carrying CFTR sequence were established. Milk of Tg females contain CFTR bound to membrane of fat globule.

17. Harvard scientist made a genitically engineered, OncoMouse or Harvard mouse that promotes the development of human cancers.
GSK scientists engineered the human mitocondrial transporter proteins “uncoupling protein -3” (UPC) in skeletal muscle in mice.
Development of first mice to produce a human drug, tPA in 1987.

18. Transgenic sheep :a new kind of farming
1985,Tracy, the sheep the first transgenic animal to produce a recombinant drug in her milk.
Tracy
Alpha 1- antitrypsin (APP) treatment for emphysema & cystic fibrosis.

19. John Clark & etal produce transgenic sheep expressing human blood clotting factor IX needed by many patients with hemophilia. They place factor IX gene under the control of a piece of sheep DNA that turns on beta lactoglobulin gene in the mammary tissue. Sheep secreted factor IX into their milk.

20. Tg cow :for better milk production
.Rosie, transgenic cow, produce human protein –enriched milk at 2.4g/lt., a more nutritionally balanced product that could be given to babies or to the elder people with special nutritional or digestive needs . Milk contains human gene alpha-lactalbumin.
Transgenic cows exist that produce milk with less lactose or cholesterol .
The A. I. Virtanen Institute in Finland produced a calf with a gene that makes the substance that promotes the growth of red cells in humans.

21. Genetic research: Tg. butterfly
Transgenic fruit flies (Drosophila melanogastor) as genetic models to study the effects of genetic changes on development.
Flies are preferred :-Ease of culture, simpler genome than vertebrates.

22. Transgenic goat
As a dairy production goats are utilized all over the world. Goats are an ideal dairy species as produce large volumes of milk with high protein content & are generally accepted as a source of dietary milk. Significant expression, (2-10g/L) of recombinant proteins in their milk.

23. “Bio –steel” from goats
In 2001, scientists in Canada spliced spider genes into the cells of lactating goats. Goats began to manufacturing silk along with their milk & secrete tiny silk strands from their body. Extracting polymer strands & weaving them into thread, scientist create a light, flexible material used in medical microsutures & tennis racket strings.
Webster & Peter carrying silk gene

24. Transgenic Pigs: as organ transplant donors to humans
Transgenic pigs may provide the transplant organs needed to alleviate the shortfall. Currently, xenotransplantation is hampered by a pig protein that can cause donor rejection but research is underway to remove the pig protein & replace it with a human protein.
Transgenic pigs abnormally have high quantity of growth hormone so responsible for increased meat production..

25. Transgenic fish Advantages of fish models for biomedical research
reason are…..
1 High level of conservation of genetic program for development & fundamental processes b/w all vertebrates.
2 Fish can be cloned more easily than mammals.
3 External fertilization & development in many species allow ready access to development & manipulation.

26. The transgene injected into eggs of Atlantic salmon consist of promoter region from antifreeze protein gene of fish ocean pout; growth hormone cDNA from salmon; & termination polyadenylation signals from 3’ end of antifreeze gene from ocean pout .

28. Agricultural application
Breeding :-
Traditional breeding is time consuming, difficult task. by transgenesis it become possible to develop traits in animals in a shorter time & with more precision.
Disease resistance:-
Scientist are attempting to produce disease- resistant animals, such as influenza- resistant pigs, but a very limited no. genes are currently known.

29. Other applications Environment friendly farm animal
Phytase transgenic pigs have been developed to address the problem of manure –based environmental problem .
Ab production in transgenic animals

30. Currently produced transgenic animals
2000 transgenic sheep produced gene targeting MoCreath etal (2000)
2001, “Ecologically correct” transgenic pig Golovan etal (2001)
2002, production of biopolymer fiber from transgenic calf.
2003, transgenic cattle producing altered milk protein compound.
2005, transgenic cow resistant to bacterial infection (mastitis)

31. Pharming product currently in development
animal
Drug/protein
Use
Alpha antitrypsin
Deficiency leads to emphysema
Sheep
Treatment of hemophilia
Pig
Factor IX
Glutamic acid decarboxylase
Treatment of type1 diabetes
Goat
Treatment of HIV
Goat
Pro 542
cow
Collagen 1 & II
Tissue repair, treatment of rheumatoid arthritis
Chicken ,cow, goat
Monoclonal Ab
Other vaccine production

32. References
Hodebine Marie –Louis (2003); animal transgenesis & cloning, application of animal tansgenesis pg ( ).
Pesternike ,Glick (1999) molecular biotechnology by Glick; methodology of transgenesis ,
Gupta P.K.(2006) first edition; Rastogi publication; Biotechnology & Genomics; transfection method & transgenic animals; ( ).
Singh B.D. (2005), second edition;Kalyani publication; Biotechnology; study of transgene expression,
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33. THANKS