Simple one-week method to construct gene-targeting vectors: application to production of human knockout cell lines
Simple one-week method to construct gene-targeting vectors: application to production of human knockout cell lines
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DOI：
10.2144/000112233
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发表时间：
2006-09-01
期刊：
影响因子：
2.7
通讯作者：
Koyama, Hideki
中科院分区：
文献类型：
--
作者：
Iiizumi, Susumu;Nomura, Yuji;Koyama, Hideki
靶向基因破坏是研究细胞和动物基因功能的有力工具。此外，这项技术还具有纠正致病突变的潜力。然而，构建靶向载体是基因靶向策略中费力的步骤，即使除了哺乳动物中同源重组的低效率之外。在这里，我们介绍了一种快速和简化的方法来构建靶向载体。该方法基于市售的MultiSite Gateway（R）技术。唯一的关键步骤是设计引物以PCR扩增同源DNA臂的基因组片段，之后根本不需要连接反应或广泛的限制性作图。因此，该方法很容易适用于胚胎干（ES）细胞的研究，以及所有的生物体的基因组已被测序。最近，我们和其他人已经表明，人类preB细胞系Nalm-6允许高效的基因靶向。简化的载体构建系统和Nalm-6细胞系中的高效基因靶向的组合使得能够在一个月内快速破坏人类基因组的几乎任何基因座，并且在2-3个月内产生缺乏感兴趣的人类基因的纯合敲除克隆。因此，我们的系统大大方便了反向遗传学的研究，特别是人类的基因。
Targeted gene disruption is a powerful tool for studying gene function in cells and animals. In addition, this technology includes a potential to correct disease-causing mutations. However, constructing targeting vectors is a laborious step in the gene-targeting strategy, even apart from the low efficiency of homologous recombination in mammals. Here, we introduce a quick and simplified method to construct targeting vectors. This method is based on the commercially available MultiSite Gateway (R) technology. The sole critical step is to design primers to PCR amplify genomic fragments for homologous DNA arms, after which neither ligation reaction nor extensive restriction mapping is necessary at all. The method therefore is readily applicable to embryonic stem (ES) cell studies as well as all organisms whose genome has been sequenced. Recently, we and others have shown that the human preB cell line Nalm-6 allows for high-efficiency gene targeting. The combination of the simplified vector construction system and the high-efficiency gene targeting in the Nalm-6 cell line has enabled rapid disruption of virtually any locus of the human genome within one month, and homozygous knockout clones lacking a human gene of interest call be created within 2-3 months. Thus, our system greatly facilitates reverse genetic studies of mammalian-particularly human-genes.