A conditional knockout resource for the genome-wide study of mouse gene function.
A conditional knockout resource for the genome-wide study of mouse gene function.
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DOI：
10.1038/nature10163
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发表时间：
2011-06-15
期刊：
影响因子：
64.8
通讯作者：
Bradley A
中科院分区：
文献类型：
--
作者：
Skarnes WC;Rosen B;West AP;Koutsourakis M;Bushell W;Iyer V;Mujica AO;Thomas M;Harrow J;Cox T;Jackson D;Severin J;Biggs P;Fu J;Nefedov M;de Jong PJ;Stewart AF;Bradley A
胚胎干细胞中的基因打靶已成为操作小鼠基因组的主要技术，在等位基因设计方面具有无与伦比的准确性，并可实现条件性诱变。为了将这些优势带给更广泛的研究群体，大规模的小鼠敲除计划正在为所有蛋白质编码基因产生一种永久性的靶向突变资源。在此我们报道了一种用于产生带有报告基因标签的条件性等位基因的高通量基因打靶流程的建立。计算等位基因设计、96孔模块化载体构建以及高效基因打靶策略相结合，以前所未有的规模对基因进行突变。到目前为止，已经在具有高度生殖系能力的C57BL/6N胚胎干细胞中产生了12000多个载体和9000个条件性靶向等位基因。本研究突出的高通量基因组工程广泛适用于大鼠和人类干细胞，并为未来旨在解读哺乳动物基因组所编码的所有基因功能的全基因组研究奠定了基础。
Gene targeting in embryonic stem cells has become the principal technology for manipulation of the mouse genome, offering unrivalled accuracy in allele design and access to conditional mutagenesis. To bring these advantages to the wider research community, large-scale mouse knockout programmes are producing a permanent resource of targeted mutations in all protein-coding genes. Here we report the establishment of a high-throughput gene-targeting pipeline for the generation of reporter-tagged, conditional alleles. Computational allele design, 96-well modular vector construction and high-efficiency gene-targeting strategies have been combined to mutate genes on an unprecedented scale. So far, more than 12,000 vectors and 9,000 conditional targeted alleles have been produced in highly germline-competent C57BL/6N embryonic stem cells. High-throughput genome engineering highlighted by this study is broadly applicable to rat and human stem cells and provides a foundation for future genome-wide efforts aimed at deciphering the function of all genes encoded by the mammalian genome.