Friedel Roland H, Soriano Philippe
Department of Neurosurgery, Mount Sinai School of Medicine, New York, USA.
Methods Enzymol. 2010;477:243-69. doi: 10.1016/S0076-6879(10)77013-0.
Gene trapping in mouse embryonic stem (ES) cells is an efficient method for the mutagenesis of the mammalian genome. Insertion of a gene trap vector disrupts gene function, reports gene expression, and provides a convenient tag for the identification of the insertion site. The trap vector can be delivered to ES cells by electroporation of a plasmid, by retroviral infection, or by transposon-mediated insertion. Recent developments in trapping technology involve the utilization of site-specific recombination sites, which allow the induced modification of trap alleles in vitro and in vivo. Gene trapping strategies have also been successfully developed to screen for genes that are acting in specific biological pathways. In this chapter, we review different applications of gene trapping, and we provide detailed experimental protocols for gene trapping in ES cells by retroviral and transposon gene trap vectors.
小鼠胚胎干细胞(ES细胞)中的基因捕获是一种对哺乳动物基因组进行诱变的有效方法。基因捕获载体的插入会破坏基因功能、报告基因表达,并为鉴定插入位点提供一个便利的标签。可以通过质粒电穿孔、逆转录病毒感染或转座子介导的插入将捕获载体导入ES细胞。捕获技术的最新进展涉及位点特异性重组位点的利用,这使得能够在体外和体内对捕获等位基因进行诱导修饰。基因捕获策略也已成功开发出来,用于筛选在特定生物学途径中起作用的基因。在本章中,我们回顾了基因捕获的不同应用,并提供了通过逆转录病毒和转座子基因捕获载体在ES细胞中进行基因捕获的详细实验方案。