Dickins Ross A, Hemann Michael T, Zilfou Jack T, Simpson David R, Ibarra Ingrid, Hannon Gregory J, Lowe Scott W
Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.
Nat Genet. 2005 Nov;37(11):1289-95. doi: 10.1038/ng1651. Epub 2005 Oct 2.
RNA interference is a powerful method for suppressing gene expression in mammalian cells. Stable knock-down can be achieved by continuous expression of synthetic short hairpin RNAs, typically from RNA polymerase III promoters. But primary microRNA transcripts, which are endogenous triggers of RNA interference, are normally synthesized by RNA polymerase II. Here we show that RNA polymerase II promoters expressing rationally designed primary microRNA-based short hairpin RNAs produce potent, stable and regulatable gene knock-down in cultured cells and in animals, even when present at a single copy in the genome. Most notably, by tightly regulating Trp53 knock-down using tetracycline-based systems, we show that cultured mouse fibroblasts can be switched between proliferative and senescent states and that tumors induced by Trp53 suppression and cooperating oncogenes regress upon re-expression of Trp53. In practice, this primary microRNA-based short hairpin RNA vector system is markedly similar to cDNA overexpression systems and is a powerful tool for studying gene function in cells and animals.
RNA干扰是一种在哺乳动物细胞中抑制基因表达的强大方法。通过通常从RNA聚合酶III启动子持续表达合成短发夹RNA，可以实现稳定的基因敲低。但是，作为RNA干扰内源性触发因素的初级微小RNA转录本通常由RNA聚合酶II合成。在此，我们表明，表达经过合理设计的基于初级微小RNA的短发夹RNA的RNA聚合酶II启动子，即使在基因组中以单拷贝存在时，也能在培养细胞和动物中产生有效、稳定且可调节的基因敲低。最值得注意的是，通过使用基于四环素的系统严格调控Trp53基因敲低，我们发现培养的小鼠成纤维细胞可以在增殖状态和衰老状态之间转换，并且由Trp53抑制和协同致癌基因诱导的肿瘤在Trp53重新表达后会消退。实际上，这种基于初级微小RNA的短发夹RNA载体系统与cDNA过表达系统非常相似，是研究细胞和动物中基因功能的强大工具。
