transgenic animals

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HOW TRANSGENIC ANIMALS ARE PRODUCED

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A SEMINAR REPORT ON TRANSGENIC ANIMALS :

A SEMINAR REPORT ON TRANSGENIC ANIMALS Submitted by:- Ranjan sahu

INTRODUCTION:

INTRODUCTION A transgenic animal is one that carries a foreign gene that has been deliberately inserted into its genome. The foreign gene is constructed using recombinant DNA methodology. In addition to the gene itself, the DNA usually includes other sequences to enable it. There are various definitions for the term transgenic animal. The Federation of European Laboratory Animal Associations defines the term as an animal in which there has been a deliberate modification of its genome, the genetic makeup of an organism responsible for inherited characteristics.

HOW ARE TRANSGENIC ANIMALS PRODUCED? :

HOW ARE TRANSGENIC ANIMALS PRODUCED? The underlying principle in the production of transgenic animals is the introduction of a foreign gene or genes into an animal (the inserted genes are called transgenes ). The foreign genes “must be transmitted through the germ line, so that every cell, including germ cells, of the animal contain the same modified genetic material.” ( Germ cells are cells whose function is to transmit genes to an organism’s offspring). To date, there are five basic methods of producing transgenic animals .

PHYSICAL TRANSFECTION:

PHYSICAL TRANSFECTION 2.1.1. DNA Microinjection The mouse was the first animal to undergo successful gene transfer using DNA microinjection. This method involves: The females are treated with hormone fertility drug to stimulate production of more eggs by the ovary. The females are allowed to mate with fertile males and fertilized eggs are collected. The donor DNA in a buffer solution is injected into one of the pronuclei (usually into the male pronucleus )

Cont…:

Cont … The two pronuclei later fuse to form the zygote which are cultured in vitro to developed up to morula or blastula stage . The developing embryos are then transferred to the uterus of one or more foster(surrogate) mothers. The embryos develop for full term through normal pregnancy and offsprings are produced. The offsprings are screened for the presence of desired gene. Animals produced by this technique are rabbits,pigs,sheeps,cows,fishes,etc . Best success achieved till now only in case of transgenic sheep i.e. one sheep per 20 eggs.

CHEMICAL TRANSFECTION:

CHEMICAL TRANSFECTION There are several chemical transfection techniques for animal cells but all are based on similar principles. The calcium phosphate mediated DNA uptake involves the formation of a co precipitate which is taken up by the cell in endocytosis . Some of the DNA fragments which enters the cell may reach the nucleus and integrated expression of such genes confers the transfection . The transformation frequency of calcium-phosphate method is generally low(1-2%).

RETROVIRUS-MEDIATED GENE TRANSFER:

RETROVIRUS-MEDIATED GENE TRANSFER A retrovirus is a virus that carries its genetic material in the form of RNA rather than DNA. This method involves: Retroviruses 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. Chimeras are inbred for as many as 20 generations until homozygous (carrying the desired transgene in every cell) transgenic offspring are born. The method was successfully used in 1974 when a simian virus was inserted into mice embryos, resulting in mice carrying this DNA.

RETROVIRUS-MEDIATED GENE TRANSFER:

RETROVIRUS-MEDIATED GENE TRANSFER

ELECTROPHORATION:

ELECTROPHORATION In this approach, transfection mixture containing cells and DNA is exposed for a very brief period (few milliseconds) to a very high voltage gradient (4000-8000v/cm). This induces transient pores in the cell membrane through which DNA seems to enter the cells. Treatment of cells with colcemid before there electrophoreted increase the frequency of transfection .

DNA PACKAGED INSIDE A BACTERIUM:

DNA PACKAGED INSIDE A BACTERIUM Agrobacterium tumifaciens mediated transfer of DNA is a common practice in plant system. It has been shown by Kunic and co-workers (2001) that this bacterium can transfer DNA in cultured human cells. It was established in mid 1990s that several bacteria infect human cells and undergo lysis releasing plasmid in host cells .

……contd:

……contd The plasmid DNA then finds its way to the host cell nucleus, where it is integrated in the genome and expressed. Another method of DNA transfer is by conjugation , through the pillus formed by bacterial cell help in the transfer of DNA. When live bacteria are used it is necessary that the bacterium is attenuated. This is because the gene transfer system uses the natural ability of bacteria to infect eukaryotic cells the bacteria may multiply and destroy host cells.

Transgenic animal model development core:

Transgenic animal model development core TRANSGENIC MICE This transgenic method has become one of the most exciting approaches of discovering the function and interaction of genes in mammals. At the University of Washington, Nathan Shock Center , this transgenic technology is used to develop new animal models for studying genetic mechanisms of the ageing process .

Two methods used for this purpose::

Two methods used for this purpose: Method-1:(The embryonic stem cell method) Embryonic stem cells are harvested from the inner cell mass (ICM) of mouse blastocysts. They can be grown in culture and retain their full potential to produce all the cell of the mature animal, including its gametes. Method-2 : (The pronucleus method) freshly fertilized eggs are harvested before the sperm had has become a pronucleus . The male pronucleus is injected with DNA.

……contd:

……contd The pronuclei have fused to form the diploid zygote nucleus. The embryo is implanted in a pseudo pregnant foster mother. By this method a giant mouse develop from a fertilized egg transformed with an rDNA molecule containing the structural gene for human GH.

KNOCKOUT MICE:

KNOCKOUT MICE Transgenic mice that carry a knockout gene ( i.e. Gene of interest replaced by a non-functional gene) is called knockout mice . Now it is possible to select and knockout (remove) a gene and make genetic modifications in the ES cells and mouse. Various knockout mice are being used in immunological research .

KNOCK IN MICE:

KNOCK IN MICE In molecular cloning and biology, a Knock-in (or Gene knock-in) refers to a genetic engineering method that involves the insertion of a protein coding cDNA sequence at a particular locus in an organism's chromosome . Typically. this is done in mice since the technology for this process is more refined, and because mouse embryonic stem cells are easily manipulated . The difference between knock-in technology and transgenic technology is that a knock-in involves a gene inserted into a specific locus, and is a "targeted" insertion. A common use of knock-in technology is for the creation of disease models. It is a technique by which scientific investigators may study the function of the regulatory machinery (e.g. promoters) that governs the expression of the natural gene being replaced.

TRANSGENIC LIVESTOCK:

TRANSGENIC LIVESTOCK Transgenic Sheep and Goats :- A good amount of work has been done in case of sheep. Transgenic sheep are produced for b etter growth and wool production and serve as bioreactors in pharmaceutical industries. Scients of Scotland cloned the sheep polly and molly in July 1997 which were transgenic, carrying human protein gene in them. Now it is used to produced a important human Protein α -1-antitrypsin(AAT). Frst transgenic sheep to produce AAT was Tracy.

TRANSGENIC CHICKENS:-:

TRANSGENIC CHICKENS:- Chickens grow faster than sheep and goats and large numbers can be grown in close quarters ; Synthesize several grams of protein in the "white" portion of their eggs. Two methods have succeeded in producing chickens carrying and expressing foreign genes. Infecting embryos with a viral vector Carrying the human gene for a therapeutic Protein. Transforming rooster sperm with a human gene and the appropriate promoters and checking for any transgenic offspring.

TRANSGENIC PIGS:

TRANSGENIC PIGS Transgenic pigs have also been produced by fertilizing normal eggs with sperm cells that have incorporated foreign DNA. This procedure, called sperm-mediated gene transfer (SMGT) may someday be able to produce transgenic pigs that can serve as a source of transplanted organs for humans .

TRANSGENIC FISH:

TRANSGENIC FISH Aquatic animals are being engineered to increase aquaculture production, for medical and industrial research, and for ornamental reasons. Production of transgenic fish is relatively easier and more successful than any other animals. Gene transfer has been done in fish like salmon, cat fish, common carp, Ran bow trout, Gold fish and Zebra fish.

TRANSGENIC MOUSE:

TRANSGENIC MOUSE Mouse is the most suitable experimental animal for study of gene transfer. The human haemoglobin gene and immunoglobin gene have also been introduced to mouse which expressed normally and the corresponding proteins were found in the blood serum .

TRANSGENIC CAT:

TRANSGENIC CAT Recently South Korean scientist produced transgenic white Turkish angora cats to glow green under ultraviolet light. These cat contain a fluorescent gene for flu protein and expressed under skin. Subsequently they produced a number of cloned cats from the skin cells of transformed mother cat. They proposed that such cat could be beneficial in diagnosis of genetic diseases and also showed a way to produced endangered animal by cloning.

Advantages of transgenic animals:

Advantages of transgenic animals Biological products:- Transgenic animals that produce useful biological products can be created by the interaction of the portion of DNA which codes for a particular product such as human protein (α-1 antitrypsin) use to treat emphysema tissue plasmogen activator (goat), blood clotting factor-VIII and IX (sheep) and Lactoferrin ( cow). Study of disease:- Many transgenic animals are developed to increase our understanding of how genes contribute to the development of disease. So that investigation of new treatments for diseases is made possible.

Agricultural Applications :-:

Agricultural Applications :- Quality :- Transgenic cows exist that produce more milk or milk with less lactose or cholesterol, pigs and cattle that have more meat on them and sheep that grow more wool. In the past, farmers used growth hormones to spur the development of animals but this technique was problematic, especially since residue of the hormones remained in the animal product . Disease resistance:- Scientists are attempting to produce disease-resistant animals, such as influenza-resistant pigs, but a very limited number of genes are currently known to be responsible for resistance to diseases in farm animals.

Medical Applications:-:

Medical Applications:- Xenotransplantation :- Patients die every year for lack of a replacement heart, liver, or kidney. For example, about 5,000 organs are needed each year in the U.K alone. 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 and replace it with a human protein . Nutritional supplements and pharmaceuticals:- Products such as insulin, growth hormone, and blood anti-clotting factors have already been obtained from the milk of transgenic cows, sheep, or goats. Research is also underway to manufacture milk through transgenesis for treatment of debilitating diseases such as phenylketonuria ( PKU)

BIOETHICS IN PRODUCTION OF TRANSGENIC ANIMAL PRODUCTION:

BIOETHICS IN PRODUCTION OF TRANSGENIC ANIMAL PRODUCTION Besides all benefits, genetic modifications raises many ethical concerns. These refer to moral objections, about the genetic modifications brought about in animals. Because these modifications results in “unnaturalness” of this technology; for example, Transfer of human gene(e.g. blood factor IX )into the food animals(e.g. sheep, etc.) Transfer of animal genes into food plants(e.g. interferon-alpha gene into plants)which may be used by vegetarians. Transfer of genes from such animals(whose flesh is not eaten by some religious people groups)into the others that they normally eat.

FUTURE OF TRANSGENESIS:-:

FUTURE OF TRANSGENESIS :- Bioethics will decide the future of transgenesis . On one hand we see enough potentiality in transgenic animals in solving major human problems. A survey was conducted to draw up public opinion on transgenic animal research. The total percentage of citizen in favors of such research in USA is 42%, Japan 54%, and New Zealand 58%. The main reasons for opposition of people are: Production of pharmaceutical proteins from animals reduces them to mere factories. Some feel that animal should have basic rights as human beings.

CONCLUSION:

CONCLUSION Transgenic animals have been very useful in biological, biomedical and biotechnological researches and they offer unique opportunity in animal husbandry, dairy and pharmaceutical industries. However, none of the approaches has so far reached the commercial stage, which signifies the gap between promise and fulfilment. Future efforts no doubt will focus on this gap and are expected to generate valuable animal lines to enhance human welfare which enrich the quality of human life. Hence time will decide the future .

REFERENCES:

REFERENCES Cornell Chronicle, May 14, 1987, page 3. Biologists invent gun for shooting cells with DNA . Klein, TM et al (1987) High-velocity microprojectiles for delivering nucleic acids into living cells . Nature 327:70-73. Shao Jun Du et al. (1992) Growth Enhancement in Transgenic Atlantic Salmon by the Use of an "All Fish" Chimeric Growth Hormone Gene Construct. Nature Biotechnology 10, 176–181 Mattingly CJ et al (2001) Green fluorescent protein (GFP) as a marker of aryl hydrocarbon receptor ( AhR ) function in developing zebrafish ( Danio rerio ). Environ Health Perspect . 2001 Aug;109(8):845–9. Dubey R.C.biotechnology . Transgenic animals. Singh B.D. (2008) biotechnology. Transgenic animals:301-353. Internet.

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