A gene knockout (abbreviation: KO) is a genetic technique in which one of an organism's genes is made inoperative ("knocked out" of the organism). However, KO can also refer to the gene that is knocked out or the organism that carries the gene knockout. Knockout organisms or simply knockouts are used to study gene function, usually by investigating the effect of gene loss. Researchers draw inferences from the difference between the knockout organism and normal individuals.
The KO technique is essentially the opposite of a gene knockin. Knocking out two genes simultaneously in an organism is known as a double knockout (DKO). Similarly the terms triple knockout (TKO) and quadruple knockouts (QKO) are used to describe three or four knocked out genes, respectively. However, one needs to distinguish between heterozygous and homozygous KOs. In the former, only one of two gene copies (alleles) is knocked out, in the latter both are knocked out.
Knockouts are accomplished through a variety of techniques. Originally, naturally occurring mutations were identified and then gene loss or inactivation had to be established by DNA sequencing or other methods.
The directed creation of a KO begins in the test tube with a plasmid, a bacterial artificial chromosome or other DNA construct, and proceeding to cell culture. Individual cells are genetically transfected 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.
To create knockout moss, transfection of protoplasts is the preferred method. Such transformed Physcomitrella-protoplasts directly regenerate into fertile moss plants. Eight weeks after transfection, the plants can be screened for gene targeting via PCR.
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. This is done by introducing short sequences called loxP sites around the gene. These sequences will be introduced into the germ-line via the same mechanism as a knock-out. This germ-line can then be crossed to another germline containing Cre-recombinase which is a viral enzyme that can recognize these sequences, recombines them and deletes the gene flanked by these sites.
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 eliminate 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.
Gene knockin is similar to gene knockout, but it replaces a gene with another instead of deleting it.
Knockout organisms are also used as screening tools in the development of drugs, to target specific biological processes or deficiencies by using a specific knockout, or to understand the mechanism of action of a drug by using a library of knockout organisms spanning the entire genome, such as in Saccharomyces cerevisiae.
- Griffiths AJ, Miller JH, Suzuki DT, Lewontin WC, Gelbart WM (2000). An Introduction to Genetic Analysis (7th ed.). New York: W. H. Freeman. ISBN 978-0-7167-3771-1.
- Reski R (June 1998). "Physcomitrella and Arabidopsis: the David and Goliath of reverse genetics". Trends in Plant Science. 3 (6): 209–210. doi:10.1016/S1360-1385(98)01257-6.
- Egener T, Granado J, Guitton M, Hohe A, Holtorf H, Lucht JM, et al. (2002). "High frequency of phenotypic deviations in Physcomitrella patens plants transformed with a gene-disruption library". BMC Plant Biology. 2 (1): 6. doi:10.1186/1471-2229-2-6.
- "YeastDeletionWebPages". Retrieved 21 February 2017.