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Knock-out

A general term (sometimes abbr. GKO, gene knock-out) relating to artificially generated null mutations (referred to also as loss-of-function mutations) of a gene.

Such mutants, which are devoid, for example, of a particular cytokine or receptor function, are created by a process called homologous recombination, targeted deletion, or targeted disruption. It essentially involves the inactivation of an endogenous fully functional gene by insertion of cloned sequences.

Principle of gene targeting involving the use of insertion or replacement type vectors. (a) Use of insertion type vectors involves a single cross-over between genomic target sequences and homologous sequences at either end of the targeting vector. The neomycin resistance gene contained within the vector serves as a positive selectable marker. (b) Gene targeting using replacement type vectors requires two cross-over events. The positive selection marker (neo) is retained while the negative selectable marker (HSV thymidine kinase) is lost. The advantage of this system is the fact that cells harboring randomly and unspecifically integrated gene constructs still carry the thymidine kinase gene. These cells can be eliminated selectively by using thymidine kinase as a selective marker. One disadvantage of the system may be the time required to handle ES cells in vitro. These cells have a certain tendency to differentiate and this can favor the subsequent generation of genetic mosaics rather than the desired germ line integration when the cells are used to create transgenic animals.

In mice the process involves manipulation of ES cells, which are used subsequently to regenerate transgenic animals carrying the genetic defect. The technique is applicable, however, to virtually all biological cell systems employed in molecular biology and cell biology.

Another way to inactivate specifically a given (cytokine) gene is the use of antisense RNA. Special techniques such as genetic ablation allow generation of animals in which only one or several cell types have been modified genetically so that they are subsequently lacking completely.

Since most cytokines have ubiquitous biological activities, their physiologic significance as normal regulators of physiology is often difficult to assess (see also: Cytokine network). Studies of gene functions and biological processes in knock-out mice and other organisms have been of particular importance in cytokine research. Unlike in vitro studies, this approach provides information about the true physiological functions of a given cytokine in vivo by studying the biological consequences of its absence. The use of cell lines lacking expression of a particular protein is instrumental also in studying potential and proposed target proteins and thus is critical for evaluating signaling mechanisms. This is because cells not expressing a particular protein may be found to be responsive or unresponsive to another protein under study.

In some instances studies with cytokine knock-out mice have revealed either that these mice develop normally and/or do not show the pronounced effects on the immune system, hematopoiesis, or other organ systems one would have expected from the known in vitro activities of the cytokines (for a good example see: IL2, IL4).

Some information about knock-out studies involving cytokine genes or their receptors may be found in as a special subentry (Transgenic /Knock-out/Antisense studies) for selected factors or cytokines.

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