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Targeted integration of DNA using mutant lox sites in embryonic stem cells

1997; Oxford University Press; Volume: 25; Issue: 4 Linguagem: Inglês

10.1093/nar/25.4.868

1362-4962

Kimi Araki, Masatake Araki, K Yamamura,

CRISPR and Genetic Engineering

Site-directed DNA integration has been achieved by using a pair of mutant lox sites, a right element (RE) mutant lox site and a left element (LE) mutant lox site [Albert et al. (1995) Plant J. , 7, 649–659], in mouse embryonic stem (ES) cells. We established ES cell lines carrying a single copy of the wild-type lox P or LE mutant lox site as a target and examined the frequency of site-specific integration of a targeting vector carrying a lox P or RE mutant lox site induced by Cre transient expression. Since our targeting vector contains a complete neo gene, random integrants can form colonies as in the case of a gene targeting event through homologous recombination. With our system, the frequency of site-specific integration via the mutant lox sites reached a maximum of 16%. In contrast, the wild-type lox P sites yielded very low frequencies (<0.5%) of site-specific integration events. This mutated lox system will be useful for 'knock-in' integration of DNA in ES cells.