Gene Knockout/mutation

An Overview

Gene knockout is a molecular biological method employed to study the function of genes by removing the gene and observing the effects on the cell or organism. It can be accomplished bydeleting the gene completely, introducing mutations into the gene, suppressing the expression of the gene, or editing the gene in the mature organism. In fact, gene knockout is a mutation that inactivates a gene function. These mutations are very helpful for traditional genetic studies as well as for modern biological techniques including functional genomics.

Figure 1. Gene knockout.

Principles of Gene Knockout/mutation

The method of gene knockout is based on gene targeting, which uses homologous recombination to modify the genome of a living organism. The introduction of defined modifications at a genomic level by gene targeting has become a widely used technique. This new method has already made a major influence, especially on the immunology.

Genomes of living organisms could be altered by gene targeting technology (targeted insertion mutagenesis) by using homologous recombination and producing a selectable and heritable genomic change that pursues a specific scientific or technological purpose. Within this process, in vitro engineered exogenous DNA sequence (i.e., plasmid, altered gene or engineered disruption cassette) is transformed into a cell in order to recombine with the targeted genomic counterpart leading to an anticipated genetic rearrangement. Therefore, the targeted genomic sequence is either changed or entirely replaced via the transforming sequence.

How Does It work?

The best way to produce a gene knockout is homologous recombination, and through gene knockout, a single gene gets deleted without effecting all other genes in an organism.

1. Knockout is made through a combination of techniques, beginning in the test tube with a plasmid, bacterial artificial chromosomes (BACs) or other DNA constructs;

2. Embryonic stem cells (ES cells) are genetically transformed and inserted into early embryos;

3. Resulting animals with the genetic change in their germline cells can then often pass the gene knockout to future generations.

Why Useful?

Gene knockout is enforceable to make a mutation into a selected gene by directly using a potentially crucial genomic clone. This approach is commonly employed in yeast genetics to assess or modify gene function, and thousands of knockouts have been obtained in mice.

Application

Gene knock-out is engineered to carry genes that have been made inoperative. These genes are called knock-out organisms or simple knock-outs, and they are utilized in assigning function to a specific gene with an unknown function that has been sequenced. Moreover, gene knock-out strategy is used to determine the target gene function via gene tagging, mutagenesis and homologous recombination and followed by researching the consequences of altered phenotype, thus produced recognizable gene function. Besides that, few knock-outs display morphological changes that indicate gene function. Large loss of function of mutants are expected to be conditional and revealed only by a specific combination of environmental conditions.