I-SceI-induced gene replacement at a natural locus in embryonic stem cells

Gene targeting is a very powerful tool for studying mammalian development and physiology and for creating models of human diseases, In many instances, however, it is desirable to study different modifications of a target gene, but this is limited by the generally low frequency of homologous recombination in mammalian cells, We have developed a novel gene-targeting strategy In mouse embryonic stem cells that is based on the induction of endogenous gap repair processes at a defined location within the genome by induction of a double-strand break (DSB) in the gene to be mutated, This strategy was used to knock in an NH2-ezrin mutant in the villin gene, which encodes an actin-binding protein expressed in the brush border of the intestine and the kidney, To induce the DSB, an I-SceI yeast meganuclease restriction site,vas first introduced by gene targeting to the villin gene, followed by transient expression of I-SceI, The repair of the ensuing DSB was achieved with high efficiency (6 x 10(-6)) by a repair shuttle vector sharing only a 2.8-kb region of homolog with the villin gene and no negative selection marker, Compared to conventional gene-targeting experiments at the villin locus, this represents a 100-fold stimulation of gene-targeting frequency, notwithstanding a much lower length of homology. This strategy will be very helpful in facilitating the targeted introduction of several types of mutations within a gene of interest.