Targeted integration of DNA using mutant lox sites in embryonic stem cells.
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 [Albertet 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 Por 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 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 loxP sites yielded very low frequencies (<0.5%) of site-specific integration events. This mutatedloxsystem will be useful for 'knock-in' integration of DNA in ES cells.
Araki
K
K
Department of Developmental Genetics, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, Japan.
Araki
M
M
Yamamura
K
K
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000235:genetics*; D004274:DNA, Recombinant / Q000235:genetics; D018274:Electroporation; D004622:Embryo, Mammalian; D051379:Mice; D016297:Mutagenesis, Site-Directed; D008249:Protein-Lysine 6-Oxidase / Q000235:genetics*; D013234:Stem Cells / Q000378:metabolism*
D004274:DNA, Recombinant; D004247:DNA; D008249:Protein-Lysine 6-Oxidase