Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector
Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector
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DOI：
10.1038/sj.embor.embor865
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发表时间：
2003-06-01
期刊：
影响因子：
7.7
通讯作者：
Clevers, H
中科院分区：
文献类型：
--
作者：
van de Wetering, M;Oving, I;Clevers, H
我们已经设计了一种多西环素调节形式的RNA聚合酶III的H1启动子，它允许通过小干扰RNA（siRNA）诱导敲低基因表达。作为原理证明，我们在结直肠癌（CRC）细胞中靶向β-连环蛋白。T细胞因子（TCF）靶基因表达由累积的β-连环蛋白诱导，并且是这些细胞中的主要转化事件。我们先前已经证明，通过显性阴性TCF的过表达破坏CRC细胞中的β-连环蛋白/TCF 4活性诱导快速的G1期阻滞和分化。我们的诱导型siRNA载体的稳定整合允许在强力霉素诱导下快速产生siRNA，随后特异性下调β-连环蛋白。在这些CRC细胞中，TCF转录因子基因活性被抑制，并发生G1期阻滞和分化。使用该载体系统对另外两个基因的抑制表明，它应该可用于基因表达的诱导性敲低。
We have designed a doxycycline-regulated form of the H1 promoter of RNA polymerase III that allows the inducible knockdown of gene expression by small interfering RNAs (siRNAs). As a proof-of-principle, we have targeted beta-catenin in colorectal cancer (CRC) cells. T-cell factor (TCF) target-gene expression is induced by accumulated beta-catenin, and is the main transforming event in these cells. We have shown previously that the disruption of beta-catenin/TCF4 activity in CRC cells by the overexpression of dominant-negative TCF induces rapid G1 arrest and differentiation. Stable integration of our inducible siRNA vector allowed the rapid production of siRNAs on doxycycline induction, followed by specific downregulation of beta-catenin. In these CRC cells, TCF reporter-gene activity was inhibited, and G1 arrest and differentiation occurred. The inhibition of two other genes using this vector system shows that it should be useful for the inducible knockdown of gene expression.