title: I-SceI-mediated double-strand break does not increase the frequency of homologous recombination at the Dct locus in mouse embryonic stem cells.
abstract: 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-SceI meganuclease embedded in the Dct genomic segment. The embryonic stem cell line was electroporated with an I-SceI expression plasmid, and a template for the DSB-repair process that carried sequence homologies to the Dct target. The I-SceI 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-SceI 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.
authors: Mouse functional Genetics, Institut Pasteur, Paris, France.
authors: Myriam
authors: M
authors: Fenina
authors: Dominique
authors: D
authors: Simon-Chazottes
authors: Sandrine
authors: S
authors: Vandormael-Pournin
authors: Jihane
authors: J
authors: Soueid
authors: Francina
authors: F
authors: Langa
authors: Michel
authors: M
authors: Cohen-Tannoudji
authors: Bruno A
authors: BA
authors: Bernard
authors: Jean-Jacques
authors: JJ
authors: Panthier
mesh_terms: D000483:Alleles; D000818:Animals; D001483:Base Sequence; D004249:DNA Damage; D017931:DNA Primers; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000378:metabolism*; D018390:Gene Targeting; D059765:Homologous Recombination; D019746:Intramolecular Oxidoreductases / Q000235:genetics*; D051379:Mice; D016133:Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D015252:Deoxyribonucleases, Type II Site-Specific; D019746:Intramolecular Oxidoreductases; C042501:dopachrome isomerase