Insulin production from Argentine cow milk clones

1. INTRODUCTION:

1.1 Insulin

Insulin is a peptide hormone made up of 51 amino acids that make up two chains A and B. Chain A has 21 amino acids and chain B has 30 amino acids. The two chains are linked by intra and inter disulfide bonds. The release of this hormone is primarily initiated by blood glucose levels.

1.2 Diabetes Mellitus

The inability of the 2 Langerhans cells to secrete enough or the inability to secrete insulin after glucose loading is called diabetes mellitus. The complications of diabetes are the formation of cataracts, acute or chronic kidney failure, heart problems, unhealed wounds, fungal infections, etc.

Due to the aforementioned complications, it has impacts on people’s standard of living. The world population of diabetics is about 200 million. WHO data reveals this will double 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 sequenced by Frederick Sanger in the 1950s. For about 60 years, diabetics relied on natural sources of insulin, with consequent problems of supply and quality. In the late 1970s and early 1980s, recombinant DNA technology enabled scientists to synthesize insulin in bacteria.

The best natural source of insulin is human insulin which can be isolated in crystalline form from the human cadaver. It costs approximately $5,000 per vial, which is practically impossible. As diabetes affects irrational sex, race and economic status, which led scientists to think of alternative techniques that would reduce the cost of production. Using plasmid vectors, scientists produce insulin from E.coli using rDNA technology. It has its own advantages and disadvantages. It has a low generation time but the chances of contamination are high.

2. TRANSGENIC ANIMALS

The generation of transgenic animals is complex both in terms of technical difficulty and ethical problems.

2.1 Use of transgenic animals to produce proteins

The use of transgenic animals to produce proteins of human interest was already in practice. One such example is the production of tissue plasminogen activator (tPA) in the milk of goats. In this case, the mammary control DNA and the DNA encoding tissue plasminogen activator are used to produce rDNA. The hybrid gene is inoculated into a fertilized ovum (isolated from a goat) by microinjection. The microinjected fertilized egg is transferred to a foster mother. The hybrid gene carriers were then mated to produce the transgenic female homozygous for the transgene. This transgenic technology allows goats to secrete tPA into their milk. A similar technique with minor modifications is used to make cows secrete insulin into their milk. 2.2 Procedure

Here the selected animal is the Jersey heifer, known for its abundant milk production. DNA from fetal Jersey heifer mammary control is isolated. In animals and plants, the ratio of DNA to protein is lower. Therefore, isolated nuclei first. This increases the DNA to protein ratio and prevents contamination of chromosomal DNA by DNA from cytoplasmic organelles. Nuclei are opened, RNA and Protein are enzymatically digested, then DNA is precipitated.

o The DNA coding for human insulin is isolated in the same way.

o It is then treated with type II restriction endonucleases to cut at specific sites.

o The necessary DNA sequences are joined by DNA ligase enzymes.

o The hybrid gene is introduced into the cell by microinjection. Once the gene enters the cell, it must enter the nucleus.

o The eggs of the Jersey heifer are taken and the nucleus is extracted with a micropipette.

o Genetically modified nuclei are fused with enucleated eggs using cloning techniques.

o The electrical stimulus causes cell division and an early embryo develops.

o Cells from the embryo are separated and implanted into surrogate mother cows.

o Gives rise to 4 genetically modified calves in 385 ± 5 days.

o These calves will reach maturity in 18 – 24 months where they will be able to produce milk.

o Once they start producing milk, insulin can be obtained by purifying and refining the milk using protein purification techniques such as HPLC.

– Scientists isolated specific cell types from the fetus of a Jersey heifer from a slaughterhouse

– The rDNA is introduced into the cell that reaches the nucleus.

– The genetically modified nuclei are fused with enucleated bovine eggs using cloning techniques.

– Electrical impulse initiates cell division

– The cells are individualized and can be implanted in 4 surrogate mother cows

– The mother cow will give birth to genetically modified calves in 385 ± 5 days

– Genetically modified calves will reach adulthood in 18 – 24 months

– Once milk production has started, insulin can be obtained by purifying and refining the milk.

3. Conclusion

o This technique can definitely reduce the production cost by at least 30%.

o Complications can be overcome by working more with this.

o This will definitely cause a revolution in the use of transgenic animals for protein production if the usual difficulties are resolved.

4. BIBLIOGRAPHY

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BIOTECHNOLOGY Mohan. P. Arora (2004) Editorial Himalaya

GENETIC ENGINEERING Desmond ST Nicholl, Paisley (2002) Cambridge University Press

MOLECULAR BIOTECHNOLOGY: Principles and Applications of Recombinant DNA Bernard R. Glick and Jack J. Pasternak (2002)