[KrutiPaneria]

Gene knockout is a genetic process by which an existing gene function can be blocked by destroying a specific gene and such organisms are called 'Knockouts'. There is a loss of function in transgenic animal in the gene knockout which is in contrast to gaining function in introducing a foreign gene. The knockout technique is carried out by incorporating a DNA sequence (usually a selectable marker) into coding region. The chromosome carrying the target gene (with four exons) with flanking sequences is subjected to homologous recombination with a vector carrying a selectable marker gene. This homologous recombination results in Gene Knockout.

Gene knockout has many applications, especially done for research purpose; it also provides information about what that gene normally does. It is also an efficient method to study about the gene that has been sequenced but hardly known for its function. The biochemical and pathological basis of human diseases can be understood by inactivating specific genes. For these purposes the researchers produced a knockout mouse which lack genes for a single organ or an organ system.

As humans share many genes with mice and mice being animal closely related to humans knockout experiments are carried out on mice. Knockout mice also offer great opportunity in development and testing of drugs and therapies. There are many examples in which knockout mice had been useful for studying different kinds of diseases like- arthritis, anxiety, cancer, diabetes, heart diseases, obesity and Parkinson disease. Knockout mouse is a genetically engineered altered mouse lacking the genes or inactivated gene for an entire organ or organ system. These mice exhibits changes in phenotypic characters as well as behavioral and biochemical characteristics. As an example of knockout mice is p53 knockout mouse, named after gene p53 coding for a protein that suppresses growth of tumors by arresting cell. The knockout mouse is usually named after the gene that has been inactivated.

The typical procedure of producing knockout mice involves several steps. First the gene intended to be knocked out is isolated from mouse gene library, followed by engineering a new DNA sequence resembling the original gene. DNA sequence should carry a marker gene which is usually not present in the normal mice’s gene. This marker gene helps in observing the changes. Simultaneously, stem cells are isolated from mouse blastocyst and grown invitro. Then by incorporation technique the DNA sequence is incorporated into the grown stem cells. By the process of homologous recombination takes place, where in altered cells will have new sequence. Now the stem cells from the unaltered cells are isolated using marker gene and incorporated into mouse blastocyst. This blastocyst now carries both original and knockout cell, and implanted into female mice uterus for development. The newborn mice is the recombinant mice exhibiting characters of both stem cells (original and knocked out stem cells) differently in various parts of body, i.e. a chimeric mouse showing patches of grey and white. When such mice is crossbred with the wild type some of the offspring would carry copy of knockout gene in all their cells, but these mice would be white in color even after being heterozygous. Further if these heterozygous offspring are interbred, the progeny would contain few offspring inheriting knockout genes from both parents carrying no functional copy of the original gene.

Limitations with knockout mice have been discussed by National Institutes of Health, which mentions that about 15% of knockout mice are developmentally lethal i.e. they do not grow to adulthood which hinders the study of genes’ function in the embryonic and adult stage. Then other limitation is that some genes are really difficult to knockout. Sometimes knocking out fails to produce an observable change in the mice or may exhibit drastically different characteristics from those observed in human despite of same gene’s inactivation. Also sometimes there are certain developmental defects and die whilst as embryos.

There are few knockout mice that have been useful in studying human health. Knockout mouse for transplantation in which liver cells are destroyed, using suicide gene, that lacks immune system. In this mouse sample human liver cells were transplanted which could develop because of lack of immune system. This way organ transplantation is made possible in animals. SCID mice were developed by eliminating a single gene and the resultant mice lost ability of producing B-lymphocytes and T-lymphocytes, from which human mouse was developed. Knockout mice with memory loss were developed by gene knockout technique, where the mice lack hippocampus – specialized area believed to memory processor in brain. Research on knockout mice for allergy is undergoing and it is expected in near future to benefit millions of sufferers of allergic reactions.