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

摘要

Site-directed DNA integration is achieved using a right element (RE) mutant lox site and a left element (LE) mutant lox site, in mouse embryonic stem (ES) cells. ES cell lines were established carrying a single copy of the wild-type loxP or LE mutant lox site as a target and examined the frequency of site-specific integration of a targeting vector carrying a loxP or RE mutant lox site induced by Cre transient expression. Since the targeting vector contains a complete neo gene, random integrants canform colonies as in the case of a gene targeting event through homologous recombination. With this system, the frequency of site-specific integration via the mutant lox sites reached a maximum of 16%. In contrast, the wild-type loxP sites yielded very low frequencies (<0.5%) of site-specific integration events. It is suggested that this mutated lox system will be useful for 'knock-in' integration of DNA in ES cells.