A large-scale, gene-driven mutagenesis approach for the functional analysis of the mouse genome
A large-scale, gene-driven mutagenesis approach for the functional analysis of the mouse genome
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DOI：
10.1073/pnas.1633296100
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发表时间：
2003-08-19
影响因子：
11.1
通讯作者：
Ruiz, P
中科院分区：
文献类型：
--
作者：
Hansen, J;Floss, T;Ruiz, P
后基因组时代的一个主要挑战是哺乳动物基因组中每个基因的功能表征。为了应对这一挑战，我们收集了小鼠胚胎干 (ES) 细胞的突变集合，这是迄今为止可公开获取的最大的此类突变集合。使用四种不同的基因陷阱载体，我们从超过 11,000 个 ES 细胞克隆中生成了与基因陷阱整合位点相邻的 5,142 个序列（基因陷阱序列标签；http://genetrap.de）。尽管大多数基因陷阱载体插入在整个基因组中随机发生，但我们发现了与载体无关的和载体特异性的整合“热点”。由于 > 50% 的热点是载体特异性的，因此我们得出结论，用基因陷阱插入使小鼠基因组饱和的最有效方法是使用基因陷阱载体的组合。当基因陷阱整合的随机样本传代至种系时，59%（29 只中的 17 只）在转基因小鼠中产生了可观察到的表型，该频率与传统基因靶向实现的频率相似。因此，基因捕获可以大规模且经济有效地生产具有分布在整个基因组中的突变的 ES 细胞克隆，这是一种可能加速基因组注释和人类疾病体内建模的资源。
A major challenge of the postgenomic era is the functional characterization of every single gene within the mammalian genome. In an effort to address this challenge, we assembled a collection of mutations in mouse embryonic stem (ES) cells, which is the largest publicly accessible collection of such mutations to date. Using four different gene-trap vectors, we generated 5,142 sequences adjacent to the gene-trap integration sites (gene-trap sequence tags; http://genetrap.de) from >11,000 ES cell clones. Although most of the gene-trap vector insertions occurred randomly throughout the genome, we found both vector-independent and vector-specific integration "hot spots." Because >50% of the hot spots were vector-specific, we conclude that the most effective way to saturate the mouse genome with gene-trap insertions is by using a combination of gene-trap vectors. When a random sample of gene-trap integrations was passaged to the germ line, 59% (17 of 29) produced an observable phenotype in transgenic mice, a frequency similar to that achieved byconventional gene targeting. Thus, gene trapping allows a large-scale and cost-effective production of ES cell clones with mutations distributed throughout the genome, a resource likely to accelerate genome annotation and the in vivo modeling of human disease.