PRODUCTION OF THERAPEUTICS FROM TRANSGENIC ANIMALS

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.

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.

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.

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.

Methods for production of transgenic animals :-

DNA microinjection. Embryonic stem cell trasfer. Targeted gene transfer. Virus mediated gene transfer.

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.

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

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.

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 .

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.

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.

Analysis of transgene integration

. 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.

Detection of mRNA expression

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.

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.

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.

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

.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.

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.

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.

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.

“Bio –steel” from goats

Webster & Peter carrying silk gene

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..

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 protein gene of fish ocean pout; growth hormone cDNA 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 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.

Environment friendly farm animal Phytase transgenic pigs have been

developed to address the problem of manure –based environmental problem .

Ab production in transgenic animals

Other applications

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)

Currently produced transgenic animals

Pharming product currently in development

animal Drug/protein Use

Sheep Alpha antitrypsin

Deficiency leads to emphysema

Pig Factor IX Treatment of hemophilia

GoatGlutamic acid decarboxylase Treatment of type1 diabetes

Goat Pro 542 Treatment of HIV

Collagen 1 & II Tissue repair, treatment of rheumatoid arthritis

cow

Chicken ,cow, goat

Monoclonal Ab Other vaccine production

References 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

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