Inducible, reversible, and stable RNA interference in mammalian cells
Inducible, reversible, and stable RNA interference in mammalian cells
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DOI：
10.1073/pnas.0306111101
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发表时间：
2004-02-17
影响因子：
11.1
通讯作者：
Mittal, V
中科院分区：
文献类型：
--
作者：
Gupta, S;Schoer, RA;Mittal, V
RNA干扰是一种有效沉默靶基因的强有力的遗传学方法。通过短发夹RNA（shRNA）的组成型表达的基因抑制的现有方法允许分析稳定沉默基因的后果，但限制了对细胞存活、细胞周期调节和细胞发育必需的基因的分析。我们已经开发了一种诱导型U6启动子，用于在人和鼠细胞中合成shRNA。含有稳定整合的shRNA表达构建体的细胞表现出严格的剂量和时间依赖性诱导动力学，在不存在诱导剂蜕皮激素的情况下具有不可检测的背景表达。正如预期的那样，胶质母细胞瘤细胞中人p53的诱导性抑制显示出惊人的形态学变化和DNA损伤引起的细胞周期停滞缺陷。值得注意的是，如通过蛋白质的再现和原始细胞表型的恢复所观察到的，诱导剂在诱导剂撤回后是可逆的。RNA干扰的诱导性和可逆性调控在哺乳动物遗传学和分子治疗学领域有着广泛的应用。
RNA interference is a powerful genetic approach for efficiently silencing target genes. The existing method of gene suppression by the constitutive expression of short hairpin RNAs (shRNAs) allows analysis of the consequences of stably silencing genes but limits the analysis of genes essential for cell survival, cell cycle regulation, and cell development. We have developed an inducible U6 promoter for synthesis of shRNAs in both human and murine cells. Cells containing stably integrated shRNA expression constructs demonstrate stringent dosage- and time-dependent kinetics of induction with undetectable background expression in the absence of the inducer ecdysone. Inducible suppression of human p53 in glioblastoma cells shows striking morphological changes and defects in cell cycle arrest caused by DNA damage, as expected. Remarkably, the inducibility is reversible after withdrawal of the inducer, as observed by reappearance of the protein and a restoration of the original cell phenotype. Inducible and reversible regulation of RNA interference has broad applications in the areas of mammalian genetics and molecular therapeutics.