REPAIR OF A SPECIFIC DOUBLE-STRAND BREAK GENERATED WITHIN A MAMMALIAN CHROMOSOME BY YEAST ENDONUCLEASE I-SCEL
REPAIR OF A SPECIFIC DOUBLE-STRAND BREAK GENERATED WITHIN A MAMMALIAN CHROMOSOME BY YEAST ENDONUCLEASE I-SCEL
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DOI：
10.1093/nar/22.25.5649
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发表时间：
1994-12-25
影响因子：
14.9
通讯作者：
WALDMAN, AS
中科院分区：
文献类型：
--
作者：
LUKACSOVICH, T;YANG, D;WALDMAN, AS
我们建立了小鼠Ltk(-)细胞系，其基因组中含有单纯疱疹病毒胸苷激酶基因(tk)，该基因已被酵母内切酶I-Scel识别序列的插入所破坏。人工引入的18 bp I-Scel识别序列可能是小鼠细胞系基因组中的独特序列。为了评估基因组 tk 基因中的诱导双链断裂 (DSB) 是否会通过基因靶向或非同源重组优先修复，我们单独使用核酸内切酶 I-Scel（两种不同基因靶向构建体之一）或将 I-Scel 与两种靶向构建体中的每一种结合电穿孔小鼠细胞系。原则上，每种靶向构建体都能够通过同源重组纠正有缺陷的基因组 tk 序列。在存在或不存在靶向构建体的情况下，用 I-Scel 对细胞进行电穿孔后回收 tk(+) 集落。通过检测小鼠基因组中I-Scel识别序列的小缺失，我们提供了证据表明，酵母内切酶I-Scel可以将特定的DSB引入活的哺乳动物细胞的基因组中。我们进一步报道，小鼠Ltk(-)细胞基因组中的DSB优先通过非同源末端连接而不是通过与外源供体序列的靶向同源重组来修复。讨论了该系统的潜在效用。
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.