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Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae.

机译:通过使用酿酒酵母的I-SceI系统诱导哺乳动物染色体中的同源重组。

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摘要

The mitochondrial intron-encoded endonuclease I-SceI of Saccharomyces cerevisiae has an 18-bp recognition sequence and, therefore, has a very low probability of cutting DNA, even within large genomes. We demonstrate that double-strand breaks can be initiated by the I-SceI endonuclease at a predetermined location in the mouse genome and that the breaks can be repaired with a donor molecule homologous regions flanking the breaks. This induced homologous recombination is approximately 2 orders of magnitude more frequent than spontaneous homologous recombination and at least 10 times more frequent than random integration near an active promoter. As a consequence of induced homologous recombination, a heterologous novel sequence can be inserted at the site of the break. This recombination can occur at a variety of chromosomal targets in differentiated and multipotential cells. These results demonstrate homologous recombination involving chromosomal DNA by the double-strand break repair mechanism in mammals and show the usefulness of very rare cutter endonucleases, such as I-SceI, for designing genome rearrangements.
机译:酿酒酵母的线粒体内含子编码的核酸内切酶I-SceI具有18 bp的识别序列,因此即使在大型基因组中,也具有非常低的切割DNA的可能性。我们证明双链断裂可以由I-SceI核酸内切酶在小鼠基因组中的预定位置启动,并且可以用断裂旁侧的供体分子同源区域修复断裂。该诱导的同源重组的频率比自发同源重组的频率高大约2个数量级,并且比在活性启动子附近的随机整合的频率高至少10倍。作为诱导的同源重组的结果,可以将异源新序列插入到断裂位点。这种重组可以发生在分化和多能细胞中的多种染色体靶标上。这些结果证明在哺乳动物中通过双链断裂修复机制涉及染色体DNA的同源重组,并显示非常罕见的切割核酸内切酶如I-SceI在设计基因组重排中的有用性。

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