Lukacsovich T, Yang D, Waldman A S
Department of Biological Sciences, University of South Carolina, Columbia 29208.
Nucleic Acids Res. 1994 Dec 25;22(25):5649-57. doi: 10.1093/nar/22.25.5649.
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.
我们建立了一种小鼠Ltk-细胞系，其基因组中含有一个单纯疱疹病毒胸苷激酶基因（tk），该基因已因酵母内切酶I-SceI识别序列的插入而被破坏。人工引入的18bp I-SceI识别序列可能是该小鼠细胞系基因组中的独特序列。为了评估基因组tk基因中诱导的双链断裂（DSB）是否会优先通过基因靶向或非同源重组进行修复，我们分别用电穿孔法将单独的内切酶I-SceI、两种不同基因靶向构建体之一单独导入小鼠细胞系，或者将I-SceI与两种靶向构建体中的每一种结合导入。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。在用I-SceI电穿孔细胞时，无论是否存在靶向构建体，都能回收tk+菌落。通过检测小鼠基因组中I-SceI识别序列处的小缺失，我们提供了证据表明酵母内切酶I-SceI可将特定的DSB引入活的哺乳动物细胞基因组中。我们进一步报告，小鼠Ltk-细胞基因组中的DSB优先通过非同源末端连接修复，而不是通过与外源供体序列进行靶向同源重组修复。本文还讨论了该系统的潜在用途。
