A large-scale gene-trap screen for insertional mutations in developmentally regulated genes in mice.
A large-scale gene-trap screen for insertional mutations in developmentally regulated genes in mice.
复制标题
大规模基因陷阱筛选小鼠发育调控基因的插入突变。
DOI：
10.1093/genetics/139.2.889
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发表时间：
1995
期刊：
影响因子：
3.3
通讯作者：
Auerbach,A
中科院分区：
文献类型：
--
作者：
Wurst,W;Rossant,J;Prideaux,V;Kownacka,M;Joyner,A;Hill,DP;Guillemot,F;Gasca,S;Cado,D;Auerbach,A
我们已经使用了基因陷阱载体和小鼠胚胎干细胞（ES）筛选在胚胎发生（DPC）的8.5天发育调控基因的插入突变。通过β-半乳糖苷酶报告基因表达测定，从38，730个载体插入的细胞系中，393个克隆整合破坏了活性转录单位。从这些细胞系中，回收290个克隆并注射到囊胚中以测定8.5-dpc嵌合小鼠胚胎中的报告基因表达。其中，279个克隆提供了足够数量的嵌合体胚胎用于分析。36例（13%）显示出限制性的表达模式，88例（32%）显示出广泛的表达，155例（55%）未显示出可检测的表达水平。进一步的分析表明，在8.5 dpc时不表达可检测水平的报告基因的克隆中，约三分之一在12.5 dpc时显示报告基因活性。因此，在胚胎干细胞中表达的大部分基因在胚胎发生过程中受到时间或空间调控。这些结果表明，胚胎干细胞中的基因陷阱诱变提供了一种有效的方法，用于分离大量发育调控基因中的突变。
We have used a gene-trap vector and mouse embryonic stem (ES) cells to screen for insertional mutations in genes developmentally regulated at 8.5 days of embryogenesis (dpc). From 38,730 cell lines with vector insertions, 393 clonal integrations had disrupted active transcription units, as assayed by beta-galactosidase reporter gene expression. From these lines, 290 clones were recovered and injected into blastocysts to assay for reporter gene expression in 8.5-dpc chimeric mouse embryos. Of these, 279 clones provided a sufficient number of chimeric embryos for analysis. Thirty-six (13%) showed restricted patterns of reporter-gene expression, 88 (32%) showed widespread expression and 155 (55%) failed to show detectable levels of expression. Further analysis showed that approximately one-third of the clones that did not express detectable levels of the reporter gene at 8.5 dpc displayed reporter gene activity at 12.5 dpc. Thus, a large proportion of the genes that are expressed in ES cells are either temporally or spatially regulated during embryogenesis. These results indicate that gene-trap mutageneses in embryonic stem cells provide an effective approach for isolating mutations in a large number of developmentally regulated genes.