Repair of a specific double-strand break generated within a mammalian chromosome by yeast endonuclease I-SceI.
We established a mouse Ltk- cell line that contains within its genome a herpes simplex virus thymidine kinase gene (tk) that had been disrupted by the insertion of the recognition sequence for yeast endonuclease I-SceI. The artificially introduced 18 bp I-SceI recognition sequence was likely a unique sequence in the genome of the mouse cell line. To assess whether an induced double-strand break (DSB) in the genomic tk gene would be repaired preferentially by gene targeting or non-homologous recombination, we electroporated the mouse cell line with endonuclease I-SceI alone, one of two different gene targeting constructs alone, or with I-SceI in conjunction with each of the two targeting constructs. Each targeting construct was, in principle, capable of correcting the defective genomic tk sequence via homologous recombination. tk+ colonies were recovered following electroporation of cells with I-SceI in the presence or absence of a targeting construct. Through the detection of small deletions at the I-SceI recognition sequence in the mouse genome, we present evidence that a specific DSB can be introduced into the genome of a living mammalian cell by yeast endonuclease I-SceI. We further report that a DSB in the genome of a mouse Ltk- cell is repaired preferentially by non-homologous end-joining rather than by targeted homologous recombination with an exogenous donor sequence. The potential utility of this system is discussed.
Department of Biological Sciences, University of South Carolina, Columbia 29208.
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Lukacsovich
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Yang
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Waldman
D000818:Animals; D001483:Base Sequence; D004260:DNA Repair; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D005796:Genes; D066298:In Vitro Techniques; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D013937:Thymidine Kinase / Q000235:genetics
D009838:Oligodeoxyribonucleotides; D029701:Saccharomyces cerevisiae Proteins; D013937:Thymidine Kinase; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific