Functional genomics in mice by tagged sequence mutagenesis
Functional genomics in mice by tagged sequence mutagenesis
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DOI：
10.1038/ng0897-338
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发表时间：
1997-08-01
期刊：
影响因子：
30.8
通讯作者：
Ruley, HE
中科院分区：
文献类型：
--
作者：
Hicks, GG;Shi, EG;Ruley, HE
大多数哺乳动物基因很快就会被定性为 cDNA 序列，而关于其功能的信息却很少。为了利用该序列信息进行大规模功能研究，开发了一种基因捕获逆转录病毒穿梭载体来破坏小鼠胚胎干（ES）细胞中表达的基因。分离了突变克隆文库，对与 400 个独立原病毒插入片段相邻的基因组 DMA 区域进行了克隆和测序。侧翼序列被称为“启动子近端序列标签”或 PST，鉴定出 63 个因病毒整合而被破坏的特定基因和匿名 cDNA。标记序列诱变的效率表明，ES 细胞中表达的 10,000-20,000 个基因中的许多基因都可以作为目标，为体内基因功能分析提供明确的突变。此外，PST 提供了第一个源自基因组 DNA 的表达序列标签，并定义了 cDNA 序列中缺失的基因特征，例如外显子边界和启动子。
Most mammalian genes will soon be characterized as cDNA sequences with little information about their function. To utilize this sequence information for large-scale functional studies,a gene trap retrovirus shuttle vector has been developed to disrupt genes expressed in murine embryonic stem (ES) cells. A library of mutant clones was isolated, and regions of genomic DMA adjacent to 400 independent provirus inserts were cloned and sequenced. The flanking sequences, designated 'promoter-proximal sequence tags', or PSTs, identified 63 specific genes and anonymous cDNAs disrupted as a result of virus integration. The efficiency of tagged sequence mutagenesis suggests that many of the 10,000-20,000 genes expressed in ES cells can be targeted, providing defined mutations for the analysis of gene functions in vivo. In addition, PSTs provide the first expressed sequence tags derived from genomic DNA, and define gene features such as exon boundaries and promoters that are missing from cDNA sequences.