dsRNA-mediated gene silencing in cultured Drosophila cells:: a tissue culture model for the analysis of RNA interference
dsRNA-mediated gene silencing in cultured Drosophila cells:: a tissue culture model for the analysis of RNA interference
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DOI：
10.1016/s0378-1119(00)00224-9
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发表时间：
2000-07-11
期刊：
影响因子：
3.5
通讯作者：
Morgan, RA
中科院分区：
文献类型：
--
作者：
Caplen, NJ;Fleenor, J;Morgan, RA
RNA干扰（RNAi）是一种转录后基因沉默的形式，已在许多植物、线虫、原生动物和无脊椎动物物种中描述。RNAi的特征在于许多特征：由双链RNA（dsRNA）诱导，高度特异性，显著的效力和跨细胞边界的传播，以及靶基因的持续下调。先前的RNAi研究已经在整个生物体或其提取物中检查了这种效应;我们现在已经检查了RNAi在组织培养中的诱导。来自三个不同物种的哺乳动物细胞的筛选没有显示出dsRNA特异性下调基因表达的证据。相反，在果蝇Schneider 2（S2）细胞中观察到RNAi。当GFP瞬时或稳定表达时，转染对应于GFP的dsRNA以剂量依赖的方式抑制S2细胞中的绿色荧光蛋白（GFP）表达。这种效应是结构特异性和序列特异性的，因为：（1）当转染反义（或正义）RNA时，几乎没有或没有观察到效应;（2）不相关的dsRNA不降低GFP表达，以及（3）对应于gfp的dsRNA对不相关的靶转基因的表达没有影响。这种无脊椎动物组织培养模型应该允许在明确定义的细胞系统中进行功能丧失的简易测定，并有助于进一步理解RNAi的机制和参与该过程的基因。(C)2000 Elsevier Science B. V.保留所有权利。
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 downregulation 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. (C) 2000 Elsevier Science B.V. All rights reserved.