Gene knockout

A gene knockout is a genetic technique in which an organism is engineered to carry genes that have been made inoperative (have been "knocked out" of the organism). This is done for research purposes. Also known as knockout organisms or simply knockouts, they are used in learning about a gene that has been sequenced, but which has an unknown or incompletely known function. Researchers draw inferences from the difference between the knockout organism and normal individuals.

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The term also refers to the process of creating such an organism, as in "knocking out" a gene.

Method

Knockout is accomplished through a combination of techniques, beginning in the test tube with a plasmid, a bacterial artificial chromosome or other DNA construct, and proceeding to cell culture. Individual cells are genetically transformed with the DNA construct. Often the goal is to create a transgenic animal that has the altered gene. If so, embryonic stem cells are genetically transformed and inserted into early embryos. Resulting animals with the genetic change in their germline cells can then often pass the gene knockout to future generations.

The construct is engineered to recombine with the target gene, which is accomplished by incorporating sequences from the gene itself into the construct. Recombination then occurs in the region of that sequence within the gene, resulting in the insertion of a foreign sequence to disrupt the gene. With its sequence interrupted, the altered gene in most cases will be translated into a nonfunctional protein, if it is translated at all.

A conditional knockout allows gene deletion in a tissue or time specific manner.

Because the desired type of DNA recombination is a rare event in the case of most cells and most constructs, the foreign sequence chosen for insertion usually includes a reporter. This enables easy selection of cells or individuals in which knockout was successful. Sometimes the DNA construct inserts into a chromosome without the desired homologous recombination with the target gene. To avoid isolation of such cells, the DNA construct often contains a second region of DNA that allows such cells to be identified and discarded.

In diploid organisms, which contain two alleles for most genes, and may as well contain several related genes that collaborate in the same role, additional rounds of transformation and selection are performed until every targeted gene is knocked out. Selective breeding may be required to produce homozygous knockout animals.

Knock-in is similar to knock-out, but instead it replaces a gene with another instead of deleting it.

See also

• Knockdown
• Knockout mouse
• Knockdown mouse
• Germline
• Gene silencing
• Planned extinction