Gupta Sunita, Schoer Rebecca A, Egan James E, Hannon Gregory J, Mittal Vivek
Cancer Genome Research Center, Cold Spring Harbor Laboratory, 500 Sunnyside Boulevard, Woodbury, NY 11797, USA.
Proc Natl Acad Sci U S A. 2004 Feb 17;101(7):1927-32. doi: 10.1073/pnas.0306111101. Epub 2004 Feb 4.
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.
RNA干扰是一种有效沉默靶基因的强大遗传学方法。通过组成型表达短发夹RNA（shRNA）来抑制基因的现有方法，能够分析稳定沉默基因的后果，但限制了对细胞存活、细胞周期调控和细胞发育所必需基因的分析。我们开发了一种可诱导的U6启动子，用于在人和小鼠细胞中合成shRNA。含有稳定整合的shRNA表达构建体的细胞，在没有诱导剂蜕皮激素的情况下，表现出严格的剂量和时间依赖性诱导动力学，背景表达不可检测。如预期的那样，在胶质母细胞瘤细胞中对人p53的可诱导抑制显示出由DNA损伤引起的显著形态变化和细胞周期停滞缺陷。值得注意的是，在撤去诱导剂后，诱导性是可逆的，这可通过蛋白质的重新出现和原始细胞表型的恢复来观察到。RNA干扰的可诱导和可逆调控在哺乳动物遗传学和分子治疗领域具有广泛的应用。
