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Targeted induction of double-strand breaks (DSBs) at natural endogenous loci was shown to increase the rate of gene replacement by homologous recombination in mouse embryonic stem cells. The gene encoding dopachrome tautomerase ( Dct ) is specifically expressed in melanocytes and their precursors. To construct a genetic tool allowing the replacement of  Dct  gene by any gene of interest, we generated an embryonic stem cell line carrying the recognition site for the yeast I- Sce I meganuclease embedded in the  Dct  genomic segment. The embryonic stem cell line was electroporated with an I- Sce I expression plasmid, and a template for the DSB-repair process that carried sequence homologies to the  Dct  target. The I- Sce I meganuclease was indeed able to introduce a DSB at the  Dct  locus in live embryonic stem cells. However, the level of gene targeting was not improved by the DSB induction, indicating a limited capacity of I- Sce I to mediate homologous recombination at the  Dct  locus. These data suggest that homologous recombination by meganuclease-induced DSB may be locus dependent in mammalian cells.

I-SceI-Mediated Double-Strand Break Does Not Increase the Frequency of Homologous Recombination at the Dct Locus in Mouse Embryonic Stem Cells