Rapid tagging of endogenous mouse genes by recombineering and ES cell complementation of tetraploid blastocysts.
Rapid tagging of endogenous mouse genes by recombineering and ES cell complementation of tetraploid blastocysts.
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DOI：
10.1093/nar/gnh128
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发表时间：
2004
影响因子：
14.9
通讯作者：
Dewang Zhou;Jin Ren;T. M. Ryan;N. Higgins;T. Townes
中科院分区：
文献类型：
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作者：
Dewang Zhou;Jin Ren;T. M. Ryan;N. Higgins;T. Townes
设计用于修饰胚胎干（ES）细胞中的内源基因的敲入载体的构建以及从这些修饰的细胞产生小鼠是耗时的。从一个想法的构思到成熟小鼠的可用性，实验的时间轴至少是9个月。我们描述了一种方法，其中该时间轴通常减少到3个月。敲入载体通过重组工程随后缺口修复（GR）拯救从细菌人工染色体克隆快速构建，并且小鼠通过将遗传修饰的ES细胞注射到四倍体胚泡中而快速衍生。我们还描述了一个串联亲和纯化（TAP）/floxed标记基因质粒和GR救援质粒，可用于TAP标签任何鼠基因。重组工程和四倍体胚泡互补的组合提供了一种用于哺乳动物基因的大规模TAP标记的手段。
The construction of knockin vectors designed to modify endogenous genes in embryonic stem (ES) cells and the generation of mice from these modified cells is time consuming. The timeline of an experiment from the conception of an idea to the availability of mature mice is at least 9 months. We describe a method in which this timeline is typically reduced to 3 months. Knockin vectors are rapidly constructed from bacterial artificial chromosome clones by recombineering followed by gap-repair (GR) rescue, and mice are rapidly derived by injecting genetically modified ES cells into tetraploid blastocysts. We also describe a tandem affinity purification (TAP)/floxed marker gene plasmid and a GR rescue plasmid that can be used to TAP tag any murine gene. The combination of recombineering and tetraploid blastocyst complementation provides a means for large-scale TAP tagging of mammalian genes.