Insulin is a peptide hormone made of 51 amino acids composing two chains A and B. The A chain has 21 amino acids and the B chain has 30 amino acids. The two chains are linked by intra and inter disulphide linkages. The release of this hormone is mainly initiated by the blood glucose levels.
1.2 Diabetes Mellitus
The inability of the 2-cells of Langerhans to secrete adequate or inability to secrete insulin following the glucose load is said to be Diabetes mellitus. The complications of diabetes are Cataract formation, Acute to Chronic renal failure, Cardiac problems, unhealed wounds, Mycoses etc.
Due to the above said complications, it has impacts on the living standards of people. The worldwide diabetic population is about 200 million. The WHO data reveal that this will become doubled by 2025 ( based on 2002 data ). This implies the importance of this hormone.
1.3 History of Insulin
Baunting and Best developed the use of insulin therapy in 1921. Insulin was the first protein to be sequenced by Frederick Sanger in 1950s. For about 60 years diabetics were dependent on natural sources of insulin with attendant problems of supply and quality. In the late 1970s and early 1980s recombinant DNA technology enabled scientists to synthesis insulin in bacteria.
The best natural source of insulin is human insulin which can be isolated in crystalline form from the cadaver of human. It costs approximately about 5000$ per vial, which is practically impossible. As diabetes affects irrational of sex, race, economic status which led scientists to think of alternate techniques that will bring down the production cost. Using plasmid vectors, scientists produce insulin from E.coli by rDNA technology. It has its own advantages and disadvantages. It has low generation time but the chances for contamination are high.
2. TRANSGENIC ANIMALS
Generation of transgenic animals is complex in terms of both technical difficulty and ethical problems.
2.1 Utilisation of Transgenic Animals to Produce Proteins
The use of transgenic animals to produce the proteins of human interest was already in practice. One of such example is production of tissue Plasminogen Activator (tPA) in the milk of goats. Here the mammary control DNA and coding DNA for tissue plasminogen activator are utilized to produce rDNA. The hybrid gene is inoculated in to fertilized egg ( isolated from a goat ) by microinjection. The microinjected fertilized egg is transferred to a foster mother. Then the hybrid gene carriers were mate to produce the transgenic female homozygous for the transgene. This transgenic technology enables goats to secrete tPA in milk. A similar technique with little modifications is used to make cows secrete insulin in milk. 2.2 Procedure
Here the animal selected is Jersey heifer which is known for its abundant milk production. The mammary control DNA of Jersey heifer fetus is isolated. In animals and Plants, the DNA to Protein ratio is less. Hence the nuclei isolated first. This increases the ratio of DNA to Protein and avoids contamination of chromosomal DNA by DNA from cytoplasmic organelles. The nuclei opened , the RNA and Protein are enzymatically digested, then the DNA is precipitated.
o The coding DNA for human insulin is isolated in the same manner.
o It is then treated with type II Restriction Endonucleases to cut at specific sites.
o The required DNA sequences are joined together using DNA ligase enzymes.
o The hybrid gene is introduced in to the cell by microinjection. Once the gene enters the cell should enter the nucleus.
o The Jersey heifer's eggs are taken and the nucleus is removed using a micropipette.
o The genetically modified nuclei are fused with enucleated eggs using cloning techniques.
o The electrical stimulus cause cell dividing and an early embryo is developed.
o The embryo cells are separated and are implanted in surrogate mother cows.
o It gives rise to 4 genetically modified calves in 385 ± 5 days.
o These calves will reach maturity in 18 - 24 months at which they are capable of producing milk.
o Once they start producing milk, the insulin can be obtained by purification and refining of milk using protein purification techniques like HPLC.
- Scientists isolated the specific cell types from Jersey heifer's fetus from a slaughter house
- The rDNA is introduced in to the cell which reaches the nucleus
- The genetically modified nuclei is fused with enucleated cattle eggs using cloning techniques
- The electrical impulse starts cell division
- The cells are individualized and can be implanted into 4 surrogate mother cows
- The mother cow will give birth to genetically modified calves in 385 ± 5 days
- The genetically modified calves will reach adulthood in 18 - 24 months
- Once they start milk production, the insulin can be obtained by purification and refining of milk.
o This technique will definitely can reduce the production cost by atleast 30%.
o The complications can be overcome by further working with this.
o This will definitely cause a revolution in the utilization of transgenic animals for protein production if the usual difficulties are solved.
BIOTECHNOLOGY Mohan. P. Arora ( 2004) Himalaya Publishing House
GENETIC ENGINEERING Desmond S. T. Nicholl, Paisley ( 2002 ) Cambridge University Press
MOLECULAR BIOTECHNOLOGY- Principles and Applications of Recombinant DNA Bernard R. Glick and Jack J. Pasternak ( 2002 )