REPAIR OF A SPECIFIC DOUBLE-STRAND BREAK GENERATED WITHIN A MAMMALIAN CHROMOSOME BY YEAST ENDONUCLEASE I-SCEL

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-Scel, The artificially introduced 18 bp I-Scel 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-Scel alone, one of two different gene targeting constructs alone, or with I-Scel 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-Scel in the presence or absence of a targeting construct. Through the detection of small deletions at the I-Scel 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-Scel, 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.