Caplen N J, Fleenor J, Fire A, Morgan R A
Clinical Gene Therapy Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA.
Gene. 2000 Jul 11;252(1-2):95-105. doi: 10.1016/s0378-1119(00)00224-9.
RNA interference (RNAi) is a form of post-transcriptional gene silencing that has been described in a number of plant, nematode, protozoan, and invertebrate species. RNAi is characterized by a number of features: induction by double stranded RNA (dsRNA), a high degree of specificity, remarkable potency and spread across cell boundaries, and a sustained down-regulation of the target gene. Previous studies of RNAi have examined this effect in whole organisms or in extracts thereof; we have now examined the induction of RNAi in tissue culture. A screen of mammalian cells from three different species showed no evidence for the specific down-regulation of gene expression by dsRNA. By contrast, RNAi was observed in Drosophila Schneider 2 (S2) cells. Green fluorescent protein (GFP) expression in S2 cells was inhibited in a dose-dependent manner by transfection of dsRNA corresponding to gfp when GFP was expressed either transiently or stably. This effect was structure- and sequence-specific in that: (1) little or no effect was seen when antisense (or sense) RNA was transfected; (2) an unrelated dsRNA did not reduce GFP expression; and (3) dsRNA corresponding to gfp had no effect on the expression of an unrelated target transgene. This invertebrate tissue culture model should allow facile assays for loss of function in a well-defined cellular system and facilitate further understanding of the mechanism of RNAi and the genes involved in this process.
RNA干扰（RNAi）是一种转录后基因沉默形式，已在多种植物、线虫、原生动物和无脊椎动物物种中有所描述。RNAi具有多个特征：由双链RNA（dsRNA）诱导、高度特异性、显著的效力且能跨越细胞边界传播，以及对靶基因的持续下调。先前对RNAi的研究在整个生物体或其提取物中检测了这种效应；我们现在研究了组织培养中RNAi的诱导情况。对来自三个不同物种的哺乳动物细胞进行筛选，未发现dsRNA对基因表达有特异性下调的证据。相比之下，在果蝇施耐德2（S2）细胞中观察到了RNAi。当绿色荧光蛋白（GFP）瞬时或稳定表达时，通过转染与gfp对应的dsRNA，S2细胞中GFP的表达受到剂量依赖性抑制。这种效应具有结构和序列特异性，具体表现为：（1）转染反义（或正义）RNA时几乎没有或没有效果；（2）无关的dsRNA不会降低GFP的表达；（3）与gfp对应的dsRNA对无关的靶转基因表达没有影响。这种无脊椎动物组织培养模型应能在明确的细胞系统中方便地进行功能丧失分析，并有助于进一步了解RNAi的机制以及参与该过程的基因。
