What are the Products of Animal Cell Cultures?

Monoclonal antibodies are useful in diagnosis and treatment of several diseases. Many beneficial traits have been transferred into animal cells in vitro and transgenic animals have been produced.

Stem cell technology has aroused possibility to strengthen the knowledge for knockout animals, cell and tissue engineering and nuclear transplantation.

Besides, gene transfer is being tried in several model animals to rectify the defective genes or replace the foreign gene for desired function. Still, this branch is on cross-road.

Antisense technology has appeared as a wonderful path to biotechnology. Its allure rested in its simplicity and specificity. Hopefully, it may be an elegant approach to drug, target validation and gene function.

1. Tissue Plasminogen Activator (tPA) (e.g. Elastase):

The enzyme tPA is a protease that occurs in mammalian cells naturally. In human body proteins exist in small amount, and converts plasminogen into plasmin. Plasmin is a serine protease which is an active enzyme that degrades fibrin of the blood clot.

The tPA is the first drug which was produced in 1987 by a biotechnology company Genetech (U.S.A). By using recombinant DNA technology the tPA gene was introduced into mammalian cells (CHO or melanoma cells). The cDNA of tPA gene was inserted into the plasmid. The recombinant plasmids were inserted into mammalian cells (Fig.10.1).

The transformed cells that secreted high amount of tPA were screened from the mixed population of cells. These were cultivated in a large ferment or to get high amount of tPA.

Now, tPA is commercially produced by a mammalian cell line carrying a high expression vector (e.g. pSV2) as a fusion of mouse DHFR (dihydroxy folate reductase) gene with the tPA gene. Recombinant protein is secreted into the medium since the tPA signal sequence is cloned along with the tPA gene.

Recombinant human t-PA is now produced by recombinant DNA technology. Injected within the first hours after a heart attack, it has saved many lives by dissolving the clot blocking the coronary artery and restoring blood flow before the heart muscle becomes irreversibly damaged. It is also used for people who suffer from an ischemic stroke (i.e. a clot in the brain). But it must not be used for hemorrhagic strokes i.e. a burst blood vessel.

2. Blood factor VIII :

There are many sex-linked genetic disorders occurring naturally in human beings. One of such diseases is haemophilia, a disease that affects blood clotting. It is also an inherited disease.

In normal individuals a blood clotting factor VIII is secreted by a gene present on X- chromosome. Haemophilia A arises due to mutation i.e. individuals lacking factor VIII on X-chromosome.

Therefore, the sufferes of haemophilia A lack factor VIII. This disease occurs in one in 10,000 males who are susceptible to the effect of mutation. Current therapy of haemophilia is the transfusion of blood factor VIII into patients.

The blood factor VIII is very large and complex. It has about 25 sites to which carbohydrate molecules are attached. Using recombinant DNA technology, factor VIII has been produced from mammalian cell culture e.g. Hamster kidney cell (Fig. 10.3).

Molecular Pharming :

Sometimes, over-expression of human genes in bacteria like E. coli fails to yield a functionally active protein in humans. Because some proteins need to be post-translationally modified (phosphorylated, glycosylated, etc.) before they come in active form. Generally, bacteria lack the specific enzymes that recognize the human protein sequences that need to be modified.

Therefore, bacterial gene product will differ from the native one. To overcome this problem, certain human genes can be introduced into farm animals. Usually yeast does this job.

When these genes are expressed in the mammary glands of the animals, the post-translationally modified protein can be isolated from milk, tested for its similarity with the native human one and be developed as a pharmaceutical, if identical with human gene product.

The genes for two different human blood clotting factors (VIII and IX) have been transferred into sheep and pig regulatory sequences that cause expression in mammary tissue. After transformation of sheep or pig embryos, genetically engineered animals have been selected that produce milk with a large percentage of human blood- clotting factor.

The novel protein from transgenic animals can be isolated from the milk, purified, and marketed. Similarly, transgenic rabbits have been developed that produce human interleukin-2, which is a protein stimulating the proliferation of T-lymphocytes, the latter plays an important role in fighting some cases of cancers.