X-ray-induced mutations in mouse embryonic stem cells.
X-ray-induced mutations in mouse embryonic stem cells.
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DOI：
10.1073/pnas.95.3.1114
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发表时间：
1998-02
影响因子：
11.1
通讯作者：
James W. Thomas;C. LaMantia;T. Magnuson
中科院分区：
文献类型：
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作者：
James W. Thomas;C. LaMantia;T. Magnuson
缺失复合物由在单个位点诱导的多个染色体缺失组成，可以提供一种在模型遗传系统中跨越几厘摩的区域的功能分析的手段。本文描述了一种识别和定位小鼠中任何克隆位点缺失的策略。首先，建立了高度多态性的生殖系感受态F1（129/Sv-+Tyr+p x CAST/Ei）小鼠胚胎干细胞系。然后，进行X射线和UV诱导的诱变以确定在整个小鼠基因组中产生缺失复合物的可行性。这里报告的选择协议，诱导突变频率，细胞遗传学和广泛的分子分析的突变在X染色体连锁的次黄嘌呤磷酸核糖转移酶（Hprt）基因座和位于9号染色体上的神经细胞粘附分子（NCAM）基因座。基于PCR的多态性微卫星标记的突变分析显示，在Ncam位点观察到28 cM的缺失。荧光原位杂交与9号染色体涂料显示，一些Ncam缺失伴随着复杂的染色体重排。此外，结合微卫星标记的杂合性丢失的缺失作图揭示了一个假定的单倍不足的区域远端Ncam。这些数据表明，在小鼠胚胎干细胞中产生X射线诱导的缺失复合物是可行的。
Deletion complexes consisting of multiple chromosomal deletions induced at single loci can provide a means for functional analysis of regions spanning several centimorgans in model genetic systems. A strategy to identify and map deletions at any cloned locus in the mouse is described here. First, a highly polymorphic, germ-line competent F1(129/Sv-+Tyr+p x CAST/Ei) mouse embryonic stem cell line was established. Then, x-ray and UV-induced mutagenesis was performed to determine the feasibility of generating deletion complexes throughout the mouse genome. Reported here are the selection protocols, induced mutation frequencies, cytogenetic and extensive molecular analysis of mutations at the X-chromosome-linked hypoxanthine phosphoribosyltransferase (Hprt) locus and at the neural cell adhesion molecule (Ncam) locus located on chromosome 9. Mutation analysis with PCR-based polymorphic microsatellite markers revealed deletions of 28 cM were observed at the Ncam locus. Fluorescence in situ hybridization with a chromosome 9 paint revealed that some of the Ncam deletions were accompanied by complex chromosome rearrangements. In addition, deletion mapping in combination with loss of heterozygosity of microsatellite markers revealed a putative haploinsufficient region distal to Ncam. These data indicate that it is feasible to generate x-ray-induced deletion complexes in mouse embryonic stem cells.