Stable suppression of gene expression by RNAi in mammalian cells
Stable suppression of gene expression by RNAi in mammalian cells
复制标题
DOI：
10.1073/pnas.032652399
复制
发表时间：
2002-02-05
影响因子：
11.1
通讯作者：
Hannon, GJ
中科院分区：
文献类型：
--
作者：
Paddison, PJ;Caudy, AA;Hannon, GJ
在包括植物、秀丽隐杆线虫、果蝇和锥虫在内的多种生物中，双链RNA（dsRNA）是基因沉默的有效触发物。在几个模型系统中，这种自然反应已经发展成为研究基因功能的有力工具。RNA干扰（RNAi）作为遗传工具的使用最近已扩展到哺乳动物细胞，其可通过用模拟沉默过程的第一步中产生的那些的小的、约22-nt RNA处理来诱导。在这里，我们表明，一些培养的小鼠细胞特异性沉默基因表达后，处理与长的dsRNA（约500 nt），这种反应显示了传统的RNAi的标志，包括沉默在转录后水平和内源性生产的约22-nt的小RNA。此外，长发夹dsRNA的强制表达诱导稳定的基因沉默。产生稳定的“敲低”细胞系的能力通过使得能够检查在长时间段内发育的表型来扩展RNAi在哺乳动物细胞中的效用，并且通过在基于表型的正向遗传选择中使用RNAi来奠定基础。
In a diverse group of organisms including plants, Caenorhabditis elegans, Drosophila, and trypanosomes, double-stranded RNA (dsRNA) is a potent trigger of gene silencing. In several model systems, this natural response has been developed into a powerful tool for the investigation of gene function. Use of RNA interference (RNAi) as a genetic tool has recently been extended to mammalian cells, being inducible by treatment with small, approximate to 22-nt RNAs that mimic those produced in the first step of the silencing process. Here, we show that some cultured murine cells specifically silence gene expression upon treatment with long dsRNAs (approximate to 500 nt), This response shows hallmarks of conventional RNAi including silencing at the posttranscriptional level and the endogenous production of approximate to 22-nt small RNAs. Furthermore, enforced expression of long, hairpin dsRNAs induced stable gene silencing. The ability to create stable "knock-down" cell lines expands the utility of RNAi in mammalian cells by enabling examination of phenotypes that develop over long time periods and lays the groundwork for by using RNAi in phenotype-based, forward genetic selections.