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 construct and--for knockouts in multi-cellular organisms--ultimately fused with a stem cell from a nascent embryo .
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 interupted, the altered gene in most cases will be translated into a nonfunctional protein, if it is translated at all.
Because recombination is a rare event in the case of most cells and most constructs, the foreign sequence chosen for insertion usually is a reporter. This enables easy selection of cells or individuals in which knockout was successful.
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 targetted gene is knocked out.
Compare: knock-in
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