Inducible suppression of Fgfr2 and Survivin in ES cells using a combination of the RNA interference (RNAi) and the Cre-LoxP system
Inducible suppression of Fgfr2 and Survivin in ES cells using a combination of the RNA interference (RNAi) and the Cre-LoxP system
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DOI：
10.1093/nar/gnh083
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发表时间：
2004-06-01
影响因子：
14.9
通讯作者：
Deng, CX
中科院分区：
文献类型：
--
作者：
Coumoul, X;Li, WM;Deng, CX
RNA 干扰 (RNAi) 是一种简单而强大的工具，广泛用于研究许多物种的基因功能。最近，已经开发出使用四环素或蜕皮激素响应系统对哺乳动物细胞中的RNAi进行诱导调节，以防止与细胞生存或细胞周期进展所必需的基因持续抑制相关的潜在致死性或非生理反应。在这里，我们证明 RNAi 的诱导调节也可以通过使用 Cre-LoxP 方法来实现。我们证明，将 loxP 侧翼的新霉素盒插入 RNA 聚合酶 III 启动子（控制基于载体的 RNAi 单元）会损害启动子活性。然而，使用他莫昔芬诱导型 Cre 构建体去除 neo 盒后，RNAi 构建体的表达可以完全恢复。我们表明，该系统可以高效抑制小鼠胚胎干 (ES) 细胞中的两种内源基因 Fgfr2 和 Survivin，基因表达水平降低、细胞增殖和集落形成减少就证明了这一点。该系统提供了一种潜在重要但简单的方法来建立突变小鼠品系，以便在打开由 Cre-LoxP 系统控制的诱导开关后在特定阶段进行功能研究。
RNA interference (RNAi) is a simple and powerful tool widely used for studying gene function in a number of species. Recently, inducible regulation of RNAi in mammalian cells using either tetracycline- or ecdysone-responsive systems has been developed to prevent potential lethality or non-physiological responses associated with persistent suppression of genes that are essential for cell survival or cell cycle progression. Here we show that the inducible regulation of RNAi also can be achieved by using a Cre-LoxP approach. We demonstrate that the insertion of a loxP-flanked neomycin cassette into RNA polymerase III promoter, which controls a vector-based RNAi unit, impairs the promoter activity. However, the expression of RNAi construct can be completely restored upon the removal of the neo cassette using a tamoxifen inducible Cre construct. We show that this system works with high efficiency in suppression of two endogenous genes, Fgfr2 and Survivin, in mouse embryonic stem (ES) cells, as evidenced by the decrease of levels of gene expression, reduced cell proliferation and colony formation. This system provides a potentially important yet simple approach to establish mutant mouse strains for functional study at defined stages upon turning on the inducible switches controlled by the Cre-LoxP system.