Generation of double-knockout embryonic stem cells.

Gene inactivation in mouse embryonic stem (ES) cells usually affects a single allele that is subsequently transmitted to the mouse germline. Upon breeding to homozygosity the consequences of complete gene ablation can be studied in the context of the complete organism. In many cases, it can be useful to study the consequences of gene ablation already in ES cells, for example, when a cellular phenotype is expected. This requires both alleles of a gene to be disrupted. Besides consecutive targeting by using different selectable marker genes, homozygosity for gene disruption can also be obtained by selecting cells for duplication of (part of) the chromosome carrying the targeted allele with concomitant loss of the wild-type allele.