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首页> 外文期刊>Molecular and Cellular Biology >Analysis of Gene Targeting and Intrachromosomal Homologous Recombination Stimulated by Genomic Double-Strand Breaks in Mouse Embryonic Stem Cells
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Analysis of Gene Targeting and Intrachromosomal Homologous Recombination Stimulated by Genomic Double-Strand Breaks in Mouse Embryonic Stem Cells

机译：基因组双链断裂在小鼠胚胎干细胞中刺激基因靶向和染色体内同源重组的分析

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摘要

To investigate the effects of in vivo genomic DNA double-strand breaks on the efficiency and mechanisms of gene targeting in mouse embryonic stem cells, we have used a series of insertion and replacement vectors carrying two, one, or no genomic sites for the rare-cutting endonuclease I-SceI. These vectors were introduced into the hypoxanthine phosphoribosyltransferase (hprt) gene to produce substrates for gene-targeting (plasmid-to-chromosome) or intrachromosomal (direct repeat) homologous recombination. Recombination at the hprt locus is markedly increased following transfection with an I-SceI expression plasmid and a homologous donor plasmid (if needed). The frequency of gene targeting in clones with an I-SceI site attains a value of 1%, 5,000-fold higher than that in clones with no I-SceI site. The use of silent restriction site polymorphisms indicates that the frequencies with which donor plasmid sequences replace the target chromosomal sequences decrease with distance from the genomic break site. The frequency of intrachromosomal recombination reaches a value of 3.1%, 120-fold higher than background spontaneous recombination. Because palindromic insertions were used as polymorphic markers, a significant number of recombinants exhibit distinct genotypic sectoring among daughter cells from a single clone, suggesting the existence of heteroduplex DNA in the original recombination product.
机译：为了研究体内基因组DNA双链断裂对小鼠胚胎干细胞中基因靶向的效率和机制的影响，我们使用了一系列带有两个，一个或无基因组位点的插入和替换载体，用于罕见的切割核酸内切酶I- Sce I。将这些载体导入次黄嘌呤磷酸核糖基转移酶（ hprt ）基因中，以产生用于基因靶向（质粒到染色体）或染色体内（直接重复）同源重组的底物。用I- Sce I表达质粒和同源供体质粒（如果需要）转染后， hprt 基因座处的重组显着增加。具有I- Sce I位点的克隆中的基因靶向频率达到1％的值，比没有I- Sce I位点的克隆中的基因靶向频率高5,000倍。沉默限制性位点多态性的使用表明，供体质粒序列取代靶染色体序列的频率随距基因组断裂位点的距离而降低。染色体内重组的频率达到3.1％，比背景自发重组高120倍。由于回文插入被用作多态性标记，大量的重组子在单个克隆的子细胞之间表现出不同的基因型扇形，这表明原始重组产物中存在异源双链DNA。

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