PRODUCTION OF THERAPEUTICS FROM TRANSGENIC ANIMALS CONTENTS Reasons

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PRODUCTION OF THERAPEUTICS FROM TRANSGENIC ANIMALS

PRODUCTION OF THERAPEUTICS FROM TRANSGENIC ANIMALS

CONTENTS: Ø Ø Ø Ø Reasons for producing transgenic animals. Historical background. What is

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

Reasons for producing transgenic animals : n n Obtain fundamental information on living organisms

Reasons for producing transgenic animals : n n 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.

Historical background : Ø Ø 1970 first transgenic mice via, viral infection, but not

Historical background : Ø Ø 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.

What is a transgenic animal : n n A transgenic animal is one that

What is a transgenic animal : n n 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.

Methods for production of transgenic animals : Ø Ø DNA microinjection. Embryonic stem cell

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

DNA microinjection n Direct microinjection of a chosen gene construct into pronuclear of a

DNA microinjection n Direct microinjection of a chosen gene construct into pronuclear of a fertilized ovum. n first methods that proved to be effective in mammals. n 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. n Manipulated fertilized ovum is transferred into the oviduct of a foster mother. n A major advantage of this method is its applicability to a wide variety of species.

Embryonic stem cell – mediated gene transfer n n n n n Make your

Embryonic stem cell – mediated gene transfer n n n n n 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

Targeted gene insertion n In this method transgene integrates at the precise site where

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

Retrovirus-mediated gene transfer n n n . Earliest method for successful gene transfer in

Retrovirus-mediated gene transfer n n n . Earliest method for successful gene transfer in mammals. Virus has transfection property. Killed virus is replication defective Virus gene is replicated with transgene 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.

Detection of transgenic animals & transgene expression Identification of transgenic animals: It utilizes either

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

Analysis of transgene integration The integration of transgene into the genome is confirmed by

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.

Detection of m. RNA expression. A high purity RNA preparation is obtain from the

Detection of m. RNA 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.

Transgenic animals applications n Transgenic mice –As disease model system : --mice model for

Transgenic animals applications n 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 beta. A 4 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.

ØHarvard scientist made a genitically engineered, Onco. Mouse or Harvard mouse that promotes the

ØHarvard scientist made a genitically engineered, Onco. Mouse or Harvard mouse that promotes the development of human cancers. ØGSK scientists engineered the human mitocondrial transporter proteins “uncoupling protein -3” (UPC) in skeletal muscle in mice. ØDevelopment of first mice to produce a human drug, t. PA in 1987.

Transgenic sheep : a new kind of farming n 1985, Tracy, the sheep the

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

John Clark & etal produce transgenic sheep expressing human blood clotting factor IX needed

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.

Tg cow : for better milk production n . Rosie, transgenic cow, produce human

Tg cow : for better milk production n . Rosie, transgenic cow, produce human protein – enriched milk at 2. 4 g/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 alphalactalbumin. 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.

Genetic research: Tg. butterfly n n Transgenic fruit flies (Drosophila melanogastor) as genetic models

Genetic research: Tg. butterfly n n 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.

Transgenic goat n As a dairy production goats are utilized all over the world.

Transgenic goat n 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, (210 g/L) of recombinant proteins in their milk.

“Bio –steel” from goats In 2001, scientists in Canada spliced spider genes into the

“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

Transgenic Pigs: as organ transplant donors to humans n n Transgenic pigs may provide

Transgenic Pigs: as organ transplant donors to humans n n 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. .

Transgenic fish Advantages of fish models for biomedical research reason are…. . 1 High

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.

The transgene injected into eggs of Atlantic salmon consist of promoter region from antifreeze

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

Agricultural application Breeding : Traditional breeding is time consuming, difficult task. by transgenesis it

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. n Disease resistance: Scientist are attempting to produce disease- resistant animals, such as influenza- resistant pigs, but a very limited no. genes are currently known. n

Other applications n Environment friendly farm animal Phytase transgenic pigs have been developed to

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

Currently produced transgenic animals n n n 2000 transgenic sheep produced gene targeting Mo.

Currently produced transgenic animals n n n 2000 transgenic sheep produced gene targeting Mo. Creath 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)

Pharming product currently in development animal Sheep Pig Goat cow Chicken , cow, goat

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

References n n n Hodebine Marie –Louis (2003); animal transgenesis & cloning, application of

References n n n Hodebine Marie –Louis (2003); animal transgenesis & cloning, application of animal tansgenesis pg (153 -163). Pesternike , Glick (1999) molecular biotechnology by Glick; methodology of transgenesis , 361 -369. Gupta P. K. (2006) first edition; Rastogi publication; Biotechnology & Genomics; transfection method & transgenic animals; (257 -264). Singh B. D. (2005), second edition; Kalyani publication; Biotechnology; study of transgene expression, 226 -229 www. wikipedia. com. org