Gene insertion using zinc finger motif proteins

This is one of the newer methods that can be used to insert a gene into the genome of an organism. This method uses proteins with a zinc finger motif, which carry a similar structure to transcription factors, and restriction endonucleases.

Protein with zinc finger motif
The zinc finger motif protein is used to search for certain sequences in DNA and is always specific for a sequence of three nucleotides. When several such proteins are joined together, a molecule is formed that can only bind to one site on the entire DNA molecule.

Restriction endonucleases
Restriction endonucleases are enzymes that have the ability to cleave DNA, usually specific for a particular nucleotide sequence. There are usually only a few such sequences on a DNA molecule. By combining several proteins with a zinc finger motif and an endonuclease, we get a tool that can be used to cleave DNA at one specific location. For the purpose of this process, it is advantageous to use restriction endonucleases that leave behind so-called sticky ends, which are then easier to join.

The principle of the procedure
This can be used for both insertion of new genes and removal of genes that are unnecessary or mutated. Insertion of a new gene is achieved in this case by inserting a recombinant stretch of DNA carrying the gene into the cell along with a protein and enzyme. The new stretch of DNA is linked to the cut site via sticky ends using hydrogen bridges. The DNA polymerase then completes its own work by attaching the bases to the strand. Gene removal works on the same principle, but requires the use of two restriction endonucleases, one from each side of the gene. The excised section of DNA is then destroyed by the cell's enzymes. The DNA molecule is then rejoined via sticky ends by the same mechanism as when the new DNA section is inserted. It should be added that both restriction endonucleases must cleave the identical sequence to make the two ends complementary. The excised gene can also be replaced by another gene.

Usage
This method opens up enormous possibilities in genetic engineering and gene therapy. An example of its use is the disruption of the CCR5 gene, the product of which is necessary for the HIV virus to enter the cell. If the gene is damaged, the CCR5 receptor is produced, but the virus cannot infect the cell. A bone marrow sample is first taken from the patient, which is treated in this way and further cultured. The successfully transformed cells are then implanted back into the patient. The immune system cells resulting from the modified bone marrow will be resistant to the virus. Similar applications for other diseases or genetic disorders are essentially unlimited.

Related articles

 * Genetic Modifications
 * Genetic engineering