An efficient system to establish multiple embryonic stem cell lines carrying an inducible expression unit.
An efficient system to establish multiple embryonic stem cell lines carrying an inducible expression unit.
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DOI：
10.1093/nar/gni043
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发表时间：
2005-03-01
影响因子：
14.9
通讯作者：
Niwa H
中科院分区：
文献类型：
--
作者：
Masui S;Shimosato D;Toyooka Y;Yagi R;Takahashi K;Niwa H
小鼠胚胎干细胞（ES）在研究中的应用日益增多，强调了它们在维持多能性和指导细胞分化的分子机制研究中的重要性。尽管可调控的转基因表达系统对于在分子水平上精细分析细胞过程是必不可少的，但尚未描述用于建立携带任何这些系统的多个ES细胞系的策略。在这里，我们报告了我们的ROSA-TET系统的发展，一个有效的系统，用于建立多个ES细胞系携带四环素（Tc）可调节的转基因在Gt（ROSA）26 asSor（ROSA 26）基因座。该系统包括将携带loxP及其突变序列的构建体敲入ROSA 26基因座的步骤，随后是使用重组酶介导的盒交换反应将待Tc调节的cDNA引入基因座的交换步骤。这两个步骤都被证明可以高效地产生所需的克隆，这表明该系统可以很容易地引入任何ES细胞系，从而同时建立携带不同TC调节的cDNA的多个细胞系。我们相信，使用这个系统将大大加快使用ES细胞的分子生物学研究。
The growing use of mouse embryonic stem (ES) cells in research emphasizes their importance in studies of molecular mechanisms that maintain pluripotency and direct cellular differentiation. Although systems for regulatable transgene expression are essential for fine analysis of cellular processes at the molecular level, a strategy for the establishment of multiple ES cell lines carrying any of these systems has not yet been described. Here, we report our development of the ROSA-TET system, an effective system for the establishment of multiple ES cell lines carrying a tetracycline (Tc)-regulatable transgene at the Gt (ROSA)26asSor (ROSA26) locus. This system contains a knock-in step of a construct carrying both loxP and its mutant sequences into the ROSA26 locus, followed by a subsequent exchange step that introduces a cDNA to be Tc-regulated to the locus using the recombinase-mediated cassette exchange reaction. Both steps are demonstrated to give desired clones with high efficiency, suggesting that this system can be introduced readily into any ES cell lines, leading to the simultaneous establishment of multiple cell lines carrying different Tc-regulated cDNAs. We believe that use of this system will strongly accelerate molecular biological research using ES cells.
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