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Gene therapy is the correction of a genetic disorder by replacing a defective gene by a correct gene in the afflicted individual.

This therapy has been tried on human successfully in a number of cases. Despite the ethical issues, raised against the practice, field trials are being carried out and clinical protocols are being formulated. Beyond the doubts of many, this has all the potential of becoming the therapy of the future.

However, in case of the animals, there is no overwhelming desire to keep alive these animals, suffering from serious genetic impairments. The same technique of gene transfer can be used to improve the productive vity of animals through advanced animal breeding programmes. People have been practicing selective animal breeding for over 1000 years for producing the desirable traits in the livestock animals.

This process has been tedious and time-consuming. Now, breeders are under an increasing market pressure to produce animals, which grow faster, yield more milk, lay bigger eggs and so on. An approach that combines breeding with molecular genetics and recombinant DNA technology, has given better results within a short span of time. It involves selecting and transferring a beneficial gene to the germ line of an animal so that the gene perpetuates through generations in a stable manner.

The research and development made by amalgamating the aforementioned branches of study is known as transgenic. It is a branch of genetic engineering that encompasses the transfer of a gene from one organism to another, and integration and expression of the gene in the recipient organism in a stable manner.

The organism that receives and expresses the gene is known as the transgenic and the gene, transgenic. The entire process is known as transgenesis. Although the process may sound simple, it requires an elaborate practice, skill and knowledge in genetic engineering. It may be summed up in the following few points.

1. The identification of the beneficial character, its encoding gene and its cell location in the donor organism.

2. The isolation of the cell carrying the beneficial gene.

3. The isolation of the beneficial gene of interest.

4. Delivery of the gene into the target cell.

5. Monitoring for the integration and expression of the gene.

Gene delivery methods

Mammalian cells are complex cells with a complex genetic make-up. The delivery of a gene of interest into the target cell is the primary task. Several methods of gene delivery are available (see below). The suitable method has to be chosen based on the target cell.

1. Calcium phosphate precipitation of DNA.

2. Microinjection of eggs.

3. Electroporation.

4. Lipofection [Fusion of DNA loaded lipid vesicles (liposomes)]

5. Viral vectors.

6. Embryonic stem cell-mediated transfer.

Calcium phosphate precipitation

It is a straight forward method of DNA delivery into mammalian cells. The DNA (transgene) is precipitated with calcium phosphate [Ca₃ (P0₄)₂] and this precipitate is mixed with the cells to be transformed. The precipitated transgene is taken inside by a process known as endocytosis. The transgene then integrates into the host DNA and expresses for the desired product. These cells are cultured in a growth medium.

The percentage of transformation of the cells and integration of the transgene into the host cell genome is very low. Therefore, a suitable marker gene is tagged to the transgene before it is delivered into the target cell.

This will enable identify the transformed and integrated cells. The untransformed cells will be discarded from the culture to avoid overcrowding.

Microinjection

Mammalian cells, specifically fertilized eggs are transformed by microinjection. The classical experiment of transferring a rat growth hormone gene into the fertilized egg of mouse was successfully conducted by R. L. Brinster and others of the University of Pennsylvania in 1982.

The generation of a transgenic mouse containing a rat growth hormone gene may be summarized by the following few points (Brinster and others, 1982).

1. The eggs of a mature female mouse were removed from the oviduct at the secondary oocyte stage and fertilized in vitro.

2. The rat growth hormone gene was isolated and the promoter of a mouse metallo- thionein gene was ligated to it forming a fusion gene.

3. The fusion gene was introduced into a plasmid forming a recombinant plasmid.

4. A fertilized mouse egg was held on the tip of a micropipette by suction and the recombinant plasmid was microinjected into the male pro-nucleus with a fine glass needle under a microscope.

5. Several plasmids joined forming a concatemer, which underwent a homologous recombination with the mouse genome at a site.

6. The microinjected mouse egg was implanted into the uterus of a surrogate mother mouse made pseudo-pregnant.

7. Development of the embryos occurred and the mouse gave birth to litters, one of which was gigantic in comparison to a normal mouse.

8. The larger size of this rat was explained due to an increased synthesis of growth hormone, which stimulated growth in the mouse.

A transgenic mouse (left) produced by microinjecting the rat growth hormone gene into the fertilized egg of mouse. The one on the right is its sibling produced normally.

Electroporation

Short pulses of electric current are passed through a buffer solution containing the target cells and the DNA fragments. Transient openings are created in the plasma membrane of the cells, through which the DNA fragments enter. When the transfer is ensured, the electric circuit is disconnected.

Lipofection

Transfer of DNA fragments in liposomes (lipid vesicles) is known as lipofection. Liposomes are lipid vesicles, bound by two layers of phospholipid molecules. The DNA fragments are encapsulated by the liposomes and are treated with the target cells. The double layered phospholipid molecules fuse with the lipid bilayer of the plasma membrane and thus, deliver the contained DNA fragments into cells.

Viral vectors

Of all the methods discussed in the preceding sections, animal viruses prove to be better vectors. Several viruses like SV 40 (simian virus 40), vaccinia virus and baculovirus and retrovirus are used as vectors for transfecting mammalian cells in culture. However, retrovirus is most suitable for mammalian cell transfection, since it is not lytic unlike other viruses i.e. it does not destroy the cell it infects.

It integrates into the host cell genome and replicates along with it. It is a RNA virus. The RNA is first reverse transcribed into the DNA and this DNA then integrates into the host cell genome.

Embryonic stem ceil-mediated gene transfer

Stem cells are embryonic undifferentiated cells, which have the potentiality to differentiate into any kind of cell of the body. Such cells are tot potent in a true sense.

These cells are isolated from the inner cell mass of the early developmental stage (blast cyst) of mammals and are known as embryonic stem (ES) cells. These ES cells are cultured in an artificially enriched growth medium and transected with marker gene-tagged transgenic.

Such transected ES cells are microinjected into the blast cyst of a .recipient animal. The manipulated blast cysts are implanted into the uterus of a pseudo pregnant surrogate mother. The surrogate mother is allowed to come to term. The offspring’s are transgenic and express the transgenic both in the somatic and germ line cells. (Presently, the ES method is used

With the mouse model system, since the ES cells of the mouse are pluripotent. However, cow, pig and chick cells do not seem to be pluripotent.

Transgenic animal examples-a success story

Mice

The method of generating transgenic mice is already explained in the section 2.3.1.2. It has been used as a model system for studying many human diseases like arthritis, hypertension, Alzheimer’s disease, coronary heart disease, some cancers and several neuro-degenerative disorders.

A normal gene is substituted by its mutant form by a molecular mechanism known as gene knock-out and the disease is created in mouse. The molecular biology of the disease is studied, therapeutic molecules tested and an effective therapy formulated. Examples of diseases studied and therapy formulated in this manner include cystic fibrosis (CF), (3 -thalasemia, atherosclerosis, retinoblastoma and Duchene muscular dystrophy (DMD).

Cows

Transgenic cattle with desired traits have been produced by microinjection of fertilized eggs. This method is costly and time consuming. It takes around two years to produce a transgenic cow. Advancements in genetic engineering may change this costly method to a cost effective method in the future. The future objective would be to produce cattle, which would be disease resistant and whose milk would contain more casein and less lactose.

Pigs, Sheep and Goats

Transgenic sheep and goats seem promising to act as bioreactors for producing important compounds like blood coagulation factors VIII & IX, tissue plasminogen activator, interferon, interleukin, growth hormone and a variety of antibodies. However, transgenic pigs have many inherent problems like they are afflicted by several diseases.

When pigs were transformed with human growth hormone gene, the transgenic offspring’s did not exhibit an increased growth rate and were less fertile. Instead, pig growth hormone gene is found to have positive effects on the rate of growth.

This may prove promising to produce transgenic pigs with more flesh and less fat in future. Moreover, transgenic pigs may be the source for organs for xenotransplantation. The first transgenic pigs were created by the PPL Therapeutics in 2001 for xenotransplantations. In such pigs, the gene coding for the enzyme a-1, 3 galactosyl transferase was silenced. This enzyme is involved in the metabolism of sugar on the plasma membrane of the cell.

Transgenic primate

The first transgenic primate was a monkey ANDi (an acronym for inserted DNA, read backward) produced at the Primate Research Centre, Oregon by inserting a GFP gene into the fertilized egg through a retrovirus. It has become an important animal in genetic studies.

Birds

Transgenic birds are produced by retrovirus transfection. Birds could be made resistant to pathogenic organisms. All poultry products could be improved by decreasing the fat content. The eggs of chickens and ducks could be factories for the production of many important proteins.

Economically important birds could be made resistant to some viruses. Avian Leukosis Virus (ALV) may be taken as an example.

The viral coat protein gene (env) is transferred into a fertilized egg of the bird by a recombinant retrovirus infection. The genetically engineered egg hatches into a bird, which contains the env gene. The gene codes for the viral coat proteins, which confer immunity to the bird to ALV infection.

Fishes

Transgenic fishes have been successfully produced by microinjecting the transgene into the blastodisc (egg cytoplasm) through the micropyle with a fine glass needle. The microinjection is not as easy to carry out as in mammalian eggs, since the egg of a fish is covered by a tough and impervious chorion all around except at one place leaving an opening known as micropyle.

The first attempt at creating a transgenic fish was made in China in 1985. A copy of the growth hormone gene was inserted into the fertilized egg of the gold fish. The engineered eggs then developed into mature fishes. At least 50% of the transformed fishes grew to a size of four times to that of an untransformed fish.

Transgenic salmons have been produced by transferring sockeye salmon growth hormone gene into the eggs of pacific salmon. Salmon is the first transgenic animal produced for food purpose. Fishes are adversely affected by the temperature of the water.

Some live in the tropical waters, some in temperate and still others in cold waters, where the temperature of the water is sub-zero. The blood of the fishes living at sub-zero temperature has an anti-freeze substance that prevents the body fluid from freezing.

The anti-freeze gene of winter flounder (fish surviving in the freezing waters) is transferred into the egg of Atlantic salmon and transgenic fish generated, which also lives in the freezing waters without the fear of the body fluid freezing.

Animal pharming (farming)

Farm animals generated by transgenesis have been used as bioreactors for the production of several pharmaceutical proteins. Scientists were looking for a transgenic host, where a large volume of these proteins could be synthesized continuously.

The farm animals were selected for this work. The speculation’was that their milk could bear these synthesized proteins in a higher concentration and it struck.

Transgenic mice were generated, which over-expressed the foreign proteins and secreted them in their milk. The mechanism was to tag the transgene to the promoter derived from the casein (milk protein) coding gene. The gene construct (transgene + the promoter) was transferred in vitro to the fertilized egg of a farm animal. The transformed egg was transplanted into the uterus of a surrogate mother.

The surrogate mother was allowed to come to term and the transgenic animal’ contained the gene construct. When it grew to maturity and produced milk, it was found to contain the product of the transgene. Milk was chosen as the medium, since it contained a high concentration of the secreted product. Several important products like tissue plasminogen activator, urokinase and a-1 antitrypsin

(used for the treatment of emphysema) have been produced in the milk of farm animals.

Ethtical considerations

Transgenic organisms are created for the benefit of the mankind. Products from such organisms are already available in many countries. Despite the promising benefits, serious concerns have been voiced from a large quarter of the population as to creating these organisms.

Firstly and most importantly, these organisms should be confined and not released to the nature. If these are released to the nature, there will be an exchange of the gene pool with the wild varieties leading to a serious impairment of the genetic diversity. Secondly, we do not have enough knowledge about the safety of the products harvested from such organisms.

There are instances of viral contaminations of these products, which have forced the abandonment of some projects. Thirdly and finally, the fact that transgenic plants and animals will serve as model systems for genetic investigations, spells cruelty. Presently, there is no regulation governing the transgenic organisms.

The quest for knowledge should not be marred on one plea or the other. Research and development in this field should continue with an enforced regulation with cooperation among governmental and non-governmental agencies of a country and also countries of the world. Let the human civilization flourish and not perish.