Repair of a specific double-strand break generated within a mammalian chromosome by yeast endonuclease I-Scel

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Abstract
We established a mouse Ltk- cell line that contains within Its genome a herpes simplex virus thymldlne klnase 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 genomlc Mr 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 electroporatlon 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.