A recombineering based approach for high-throughput conditional knockout targeting vector construction.
A recombineering based approach for high-throughput conditional knockout targeting vector construction.
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DOI：
10.1093/nar/gkm163
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发表时间：
2007
影响因子：
14.9
通讯作者：
Liu P
中科院分区：
文献类型：
--
作者：
Chan W;Costantino N;Li R;Lee SC;Su Q;Melvin D;Court DL;Liu P
体内哺乳动物基因的功能分析主要通过分析敲除小鼠来实现。现在，几种哺乳动物基因组的测序已经完成，了解所有基因的功能是后基因组时代的下一个主要挑战。基因敲除突变小鼠的产生目前已经由许多研究小组实现，但只能通过逐个进行个体敲除。新的技术进步和现有技术的改进对于小鼠全基因组靶向突变至关重要。我们在这里描述了新的重组工程试剂和协议，使重组工程在96孔格式进行。因此，我们能够同时构建96个条件敲除靶向载体。我们新的重组工程系统使其成为现实，以产生大量的精确工程DNA结构的功能基因组学研究。
Functional analysis of mammalian genes in vivo is primarily achieved through analysing knockout mice. Now that the sequencing of several mammalian genomes has been completed, understanding functions of all the genes represents the next major challenge in the post-genome era. Generation of knockout mutant mice has currently been achieved by many research groups but only by making individual knockouts, one by one. New technological advances and the refinements of existing technologies are critical for genome-wide targeted mutagenesis in the mouse. We describe here new recombineering reagents and protocols that enable recombineering to be carried out in a 96-well format. Consequently, we are able to construct 96 conditional knockout targeting vectors simultaneously. Our new recombineering system makes it a reality to generate large numbers of precisely engineered DNA constructs for functional genomics studies.