A highly efficient recombineering-based method for generating conditional knockout mutations
A highly efficient recombineering-based method for generating conditional knockout mutations
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DOI：
10.1101/gr.749203
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发表时间：
2003-03-01
期刊：
影响因子：
7
通讯作者：
Copeland, NG
中科院分区：
文献类型：
--
作者：
Liu, PT;Jenkins, NA;Copeland, NG
基于噬菌体的大肠杆菌同源重组系统最近被开发出来，现在可以亚切或修饰克隆到质粒、bac或pac中的DNA，而不需要限制性内切酶或DNA连接酶。这种新形式的染色体工程，被称为重组，在功能基因组研究中有许多不同的用途。在这里，我们描述了一种新的基于重组的方法来产生条件小鼠敲除（cko）突变。该方法采用由λ噬菌体Red蛋白介导的同源重组，通过间隙修复将bac中的DNA亚切到高拷贝质粒中，并与Cre或Flpe重组酶一起将IoxP或FRT位点引入到亚切DNA中。与其他使用短的45-55-bp同源区域进行重组的方法不同，我们的方法使用更长的同源区域。我们还利用了几种新的大肠杆菌菌株，其中重组所需的蛋白质是由有缺陷的温度敏感的lambda噬菌体表达的，而Cre或Flpe重组酶是由阿拉伯糖诱导的启动子表达的。我们还描述了两种新的Neo选择盒，在大肠杆菌和小鼠胚胎干细胞中都能很好地工作。我们的方法快速、高效、可靠，可以在不到2周的时间内生成co靶向载体。这种方法还应该促进敲入突变和转基因构建的产生，以及加快对基因内或基因附近的调控元件和功能域的分析。
Phage-based Escherichia coli homologous recombination systems have recently been developed that now make it possible to subdone or modify DNA cloned into plasmids, BACs, or PACs without the need for restriction enzymes or DNA ligases. This new form of chromosome engineering, termed recombineering, has many different uses for functional genomic studies. Here we describe a new recombineering-based method for generating conditional mouse knockout (cko) mutations. This method uses homologous recombination mediated by the lambda phage Red proteins, to subdone DNA from BACs into high-copy plasmids by gap repair, and together with Cre or Flpe recombinases, to introduce IoxP or FRT sites into the subdoned DNA. Unlike other methods that use short 45-55-bp regions of homology for recombineering, our method uses much longer regions of homology. We also make use of several new E coli strains, in which the proteins required for recombination are expressed from a defective temperature-sensitive lambda prophage, and the Cre or Flpe recombinases from an arabinose-inducible promoter. We also describe two new Neo selection cassettes that work well in both E coli and mouse ES cells. Our method is fast, efficient, and reliable and makes it possible to generate cko-targeting vectors in less than 2 wk. This method should also facilitate the generation of knock-in mutations and transgene constructs, as well as expedite the analysis of regulatory elements and functional domains in or near genes.