Presented by:Rishabh Maheshwari M.Pharma (MPC) Ist year, SGSITS,Indore.

Transgenic techniques describes the process of introducing foreign deoxyribonucleic acid (DNA) into a host organism's Genome

Transgenic techniques is a technique in which an organism (typically a mouse) that is engineered to carry a foreign gene, or transgene of choice as part of its own genetic material for Disease models and human welfare.

Transgenics are genetically modified organisms with DNA from another source inserted into their genome. A large number of transgenic animals have been created such as Mice, Cows, Pigs, Sheep, Goats, Fish, and Frogs.

TRANSGENIC TECHNOLOGY

DNA microinjection.

Retrovirus-mediated gene transfer (RMGT).

Sperm-mediated gene transfer (SMGT).

Embryonic stem cell-mediated gene transfer.

METHODS USED FOR ANIMAL

In the past 20 years, DNA microinjection has become the most widely applied method for gene transfer in animals.

Introducing the transgene DNA directly into the zygote at an early stage of development. (No vector required)

This method involves:

1)Transfer of a desired gene construct (of a single gene or a combination of genes that are recombined and then cloned) from another member of the same species or from a different species into the pronucleus of a reproductive cell;

2)In vitro culture of the manipulated cells to develop to a specific embryonic phase; and

3) Then transfer of the embryonic cells to the recipient female.

1. DNA Microinjection

Microinjection into the germ line > transgenic animal

Gene injected into the male pronuclei (i.e Pronuclei injn)

2. Retrovirus-mediated gene transfer (RMGT) The word “retro” means, when the virus vectors infect a host cell, the

viral RNA is reverse transcribed in the cytoplasm making linear double-stranded DNA.

A retrovirus is a virus that carries its genetic material in the form of RNA rather than DNA.

In this method, retroviruses are used as vectors to transfer genetic material into the host cell, resulting in a chimera, an organism consisting of tissues or parts of diverse genetic constitution.

When cells are infected by retroviruses, the resultant viral DNA, after reverse transcription and integration, becomes a part of the host cell genome to be maintained for the life of the host cell.

3. Sperm-mediated gene transfer (SMGT)

SMGT is a simple and efficient technique to produce transgenic mice.

Use of “Linker protein" to attach DNA to sperm which transfer the new DNA during fertilization.

The blastocyst (inner layer of a fertilized egg) is harvested and mixed with recombinant DNA and inserted back in the blastocyst.

This method involves: Isolation of totipotent stem cells (stem cells that can

develop into any type of specialized cell) from embryos.

The desired gene is inserted into these cells.

Cells containing the desired DNA are incorporated into the host's embryo.

4. Embryonic stem cell-mediated gene transfer

In this technique, DNA was mixed with sperm cells before in vitro. 30% of offspring mouse were integrated in foreign DNA.

The basic principle of sperm-mediated gene transfer is: seminal plasma-free sperm cells are suspended in the appropriate medium, and then incubated with DNA.

The resultant DNA-carrying sperms are then used to fertilize eggs, via in vitro fertilization or artificial insemination or, in the case of aquatic animals, via waterborne (natural) fertilization.

Transgenic technology holds a great potential in different groups on the basis of benefits of these animals to human welfare can be grouped into areas:

1. Clinical Application,

2. Agricultural application,

3. Industrial application, and

4. Transgenic model (Mice).

APPLICATION OF TRANSGENIC TECHNOLOGY

(A) Models for human diseaseThe biomedical sciences rely heavily on animal models as tools for the discovery and development of therapeutic interventions. Some examples include:

Gene Therapy: Human gene therapy involves adding a normal copy of a gene (transgene) to the genome of a person carrying defective copies of the gene.

Genetic basis of human and animal disease.

Disease resistance in humans and animals.

Drug and product testing and/or screening.

1. CLINICAL APPLICATION

(B) Production of Pharmaceutical in transgenic animalsThe impact of transgenic animals on pharmaceutical development could soon expand as recombinant proteins expressed and secreted by transgenic animals move toward regulatory approval and production.

ATryn®, was the first product derived from a transgenic animal to be submitted for formal regulatory approval in Europe or the USA.

(C) Transgenic expression of immunoglobulin's

Transgenic animal producing a disease specific Ig, chimeric Igs were created by ligating the variable region exon of a previously characterized Ig to the constant region exon.

(D)Xenotransplantation Transplanted organs may be obtained from transgenic animals. E.g.

Transgenic pigs may provide the transplanted 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 and replace it with a human protein. For organ and tissue transplantation, which is known as a "species of daughter cells “

(A) BreedingTraditional cross breeding have been used for ages to create chickens, cows, pigs etc.

Farmers have always used selective breeding to produce animals that exhibit desired traits (e.g., increased milk production, high growth rate).

Traditional breeding is a time-consuming, difficult task.

2. AGRICULTURAL APPLICATION

(B) Quality Herman, a transgenic bull carries a human gene for Lactoferrin

(gene responsible for higher iron content)

Pigs and cattle that have more meat on them.

Sheep that grow more wool.

Eggs can be made healthier with high quality protein.

By extracting polymer strands from the milk and weaving them into thread, the scientists can create a light, tough, flexible material that could be used in such applications as military uniforms, medical micro sutures, and tennis racket strings.

Microorganisms have been engineered to produce a wide variety of proteins, which in turn can produce enzymes that can speed up industrial chemical reactions.

3. INDUSTRIAL APPLICATION

Genetically modified mice are the most common animal model for transgenic research. Transgenic mice are currently being utilized to study a variety of diseases including cancer, obesity, heart disease, arthritis, anxiety, and Parkinson’s disease.

The two most common types of genetically modified mice are knockout mice and oncomice .

4. TRANSGENIC MODEL (MICE)

Transgenic mice can be used both as model systems for human diseases and as test cases to determine if the production of a potential therapeutic agent is feasible.

However, a mouse or rodent is not a human being, even though it is a mammal, and thus the information gathered from transgenic rodent is not always medical relevant.

But, on the other hand, clinical insight about the etiology of a complex diseases can be discovered.

This technique is used for the treament of Alzheimer disease, arthritis, muscular dystrophy, tumorigenesis, hypertension, neurodegenerative disorders, endocrinological dysfunction, coronary disease, obesity and many others diseases.

USES OF ANIMAL MODEL

ADVANTAGES OF TRANSGENIC MODELThese animals are very useful for delineating the function of newly discovered genes as well as for producing useful proteins in large animals.

Transgenic animals are useful as disease models and producers of substances for human welfare.

Regulation of gene expression.

Transgenic animals have potentially broad application for the improvement of animal production quality, the enhancement of productivity, the studies of human disease models and the production of pharmaceuticals.

Though the field of transgenic animals has advanced considerably but few advancements are considered to be most important.• Introduction of the transgene • Transgene integration• Transgene expression• Transgene transmission• Use of embryonic stem cells• Pharmaceutical products from transgenic animals

SOME IMPORTANT ADVANCES

Many animals die or are born horribly disfigured as research is conducted.

Transgenic animals can radically change the direction of evolution, which can result in drastic consequences for nature and humans alike.

Dietary and food safety concerns: Foreign gene inserted in the chromosome locus may also result in different genetic changes in different degrees, causing unintended effects.

Environmental impacts: It may also lead to the loss of the wild life, resulting in a decline in genetic diversity.

DISADVANTAGES OF TRANSGENIC MODEL

Transgenic technology have been developed rapidly and provided more and improved platforms for the preparation of transgenic animals since their emergence.

All of these developments will provide new ideas and bring forth important changes in fields like medicine, health and livestock improvement.

In particular, the economic and social benefits from the production of bioreactors, drug production, and organ culture for human transplantation will be great.

CONCLUSION

1. Beardmore, J.A. (1997) Transgenics: autotransgenics and allotransgenics. Transgen. Res.6,107–108.

2. Polites, H.G. and Pinkert, C.A. (2002) DNA Microinjection and Transgenic Animal Production. In Transgenic Animal Technology: A Laboratory Handbook(2nd edition) (Pinkert, C.A.,ed.), pp. 15–70, Academic Press.

3. Dorling, A. and Lechler, R.I. (1997) Xenotransplantation: immune barriers beyond hyperacute rejection . Clin. Sci.93,493–505.

4. Brinster, R.L.et al.(1983) Expression of a microinjected immunoglobulin gene in the spleen of transgenic mice.Nature306, 332–336

REFERENCES

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8. Pinkert, C.A. (2002) Introduction to transgenic animal technology. In Transgenic Animal Technology: A Laboratory Handbook(2nd ed.)(Pinkert, C.A., ed.), pp. 3–12, Academic Press.