Analysis of gene targeting and intrachromosomal homologous recombination stimulated by genomic double-strand breaks in mouse embryonic stem cells
Analysis of gene targeting and intrachromosomal homologous recombination stimulated by genomic double-strand breaks in mouse embryonic stem cells
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DOI：
10.1128/mcb.18.7.4070
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发表时间：
1998-07-01
影响因子：
5.3
通讯作者：
Berg, P
中科院分区：
文献类型：
--
作者：
Donoho, G;Jasin, M;Berg, P
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打靶效率和机制的影响，我们使用了一系列携带两个、一个或没有稀有切割内切酶I - SceI基因组位点的插入和替换载体。这些载体被引入次黄嘌呤磷酸核糖转移酶（hprt）基因，以产生用于基因打靶（质粒到染色体）或染色体内（直接重复）同源重组的底物。在用I - SceI表达质粒和同源供体质粒（如果需要）转染后，hprt基因座处的重组显著增加。具有I - SceI位点的克隆中基因打靶的频率达到1%，比没有I - SceI位点的克隆高5000倍。使用沉默的限制性位点多态性表明，供体质粒序列替换目标染色体序列的频率随着与基因组断裂位点距离的增加而降低。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，大量重组体在单个克隆的子代细胞中表现出明显的基因型分区，这表明原始重组产物中存在异源双链DNA。
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.