APPLICATIONS OF BIOTECHNOLOGY- GENE THERAPY

 GENE THERAPY is done to correct gene defect in an individual. In this method normal gene for functioning is introduced into the cell to cure the defect.

The first clinical gene therapy was given in 1990 to a 4 year old girl, with adenosine deaminase (ADA) deficiency. The enzyme is required for immune system to function. The disorder in girl arose is due to deletion of the ADA gene. 

For gene therapy, lymphocytes from the blood of the patient are separated and cultured outside the body. These lymphocytes were introduced with cDNA using a retroviral vector. These genetically engineered  cells were returned to the patient. These cells are not immortal and a periodic transfer of recombinant cells is required in patients.

If the cDNA is introduced in an egg or early embryo, a permanent cure can be achieved.

APPLICATIONS OF BIOTECHNOLOGY- human Insulin

HUMAN INSULIN / GENETICALLY ENGINEERED INSULIN-

INSULIN is produced by ISLETS OF LANGERHANS CELLS OF PANCREAS.

This hormone regulates blood sugar levels.

Insulin was earlier extracted from the pancreas of slaughtered cattles and pigs. This was causing allergic reactions in some patients.

Insulin is synthesized as pro-hormone which needs to be processed to become active. Pro-insulin consists of A, C, AND B PEPTIDE CHAINS. In mature insulin, this C PEPTIDE is not present and it is removed by processing.  If the gene for insulin is introduced in BACTERIAL CELL, a mature form of insulin cannot be obtained.

In 1983, Eli Lilly, an  American company prepared two DNA sequences of A and B chains of human insulin and introduced them in E.coli  cells to produce insulin chains, separately. These chains were extracted and combined by creating disulfide bonds to form human insulin.

IMPORTANT TO NOTE: Hakura et al in 1977 chemically synthesized DNA sequence for two chains A and B and separately inserted into two pBR 322 plasmid vector.

Insulin production by recombinant DNA technology is designed by Gilbert and Villokomaroff in 1978.

BIOTECHNOLOGY- PROCESSES OF RECOMBINANT DNA TECHNOLOGY

 THE STEPS INVOLVED IN RECOMBINANT DNA TECHNOLOGY ARE;

1. ISOLATION OF DNA / GENETIC MATERIAL:

Bacterial cells can be lysed using lysozyme, plant cells can be treated with cellulase and fungal cells can be treated with chitinase to break open cells and release DNA and cell content. 

RNA can be removed by treating with RNase and proteins are removed by treating with proteases. Purified DNA can be precipitated with chilled ethanol. Cloudy, precipitated DNA can be observed in test tube.

2. CUTTING DNA USING RESTRICTION ENDONUCLEASES:

DNA from the source organism is cleaved by restriction endonuclease to create restriction fragments.

Vector DNA is also cleaved using the same restriction endonuclease.

These fragments can be separated using agarose gel electrophoresis. 

3. INSERTION OF DESIRED FOREIGN GENE INTO CLONING VECTOR:

Gene of interest is joined with vector DNA using DNA ligase. The combination of vector DNA and foreign DNA is called RECOMBINANT DNA OR CHIMERIC DNA. 

4. TRANSFER OF rDNA INTO HOST:

This can be achieved by a transformation in bacterial cells or Microinjection in animal cells or by using gene gun [ biolistics ] in plant cells.

BIOTECHNOLOGY- COMPETENT HOST

 RECOMBINANT DNA can be introduced into the bacterial cell by TRANSFORMATION. 

TRANSFORMATION is the uptake of naked DNA by bacterial cells. In order to force bacteria to take plasmid or recombinant DNA, the bacterial cells are made competent. This is done by treating bacteria with divalent cations such as calcium which increases the efficiency with which DNA enters the bacterium through the pores in its cell wall.

Recombinant DNA is incubated with bacterial cells on ice, followed by placing them briefly at 42⁰ C and again placing them on ice. This creates a heat shock and makes bacterial cells competent.

In ANIMAL cells DNA can be introduced using a method called MICROINJECTION. In this recombinant DNA is directly injected into the cell.

In PLANTS, BIOLISTICS OR GENE GUN technique is used to transfer recombinant DNA. In this technique plant cells are bombarded with micro particles of gold and tungsten coated with recombinant DNA.