Modulation of Gene Expression by Lentiviral-Mediated Delivery of Small Interfering RNA
Modulation of Gene Expression by Lentiviral-Mediated Delivery of Small Interfering RNA
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DOI：
10.4161/cc.2.3.376
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发表时间：
2003-01-01
期刊：
影响因子：
4.3
通讯作者：
Eder, Matthias
中科院分区：
文献类型：
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作者：
Scherr, Michaela;Battmer, Karin;Eder, Matthias
RNA干扰(RNAi)介导双链RNA触发的序列特异性转录后基因沉默。RNAi是线虫功能基因组学研究的有效工具，但RNAi的生化特性阻碍了其在哺乳动物细胞中的应用。在这里，我们分析了哺乳动物RNAi在不同细胞系中的动力学和定量方面，使用新型慢病毒构建，其双H1-shRNA表达盒位于LTR的U3区域。以增强型绿色荧光蛋白(EGFP)作为RNAi的靶基因，红色荧光蛋白(RFP)作为细胞内siRNA表达的替代标记，我们发现长期的siRNA表达介导了稳定的RNAi。此外，RNAi诱导的基因沉默在同种细胞内和不同细胞群体之间从最小到完全的功能丧失表型不同。有趣的是，基因沉默的程度与慢病毒的整合以及靶细胞中siRNA的表达水平有关。最后，我们展示了正常CD34(+)造血祖细胞中细胞因子受体基因的功能基因沉默。这些数据表明，使用合适的慢病毒构建物来量化活细胞中的siRNA表达，可以在多种靶细胞中以剂量依赖的方式分析功能丧失的表型。
RNA interference (RNAi) mediates sequence-specific posttranscriptional gene silencing triggered by double-stranded RNA. RNAi is an effective tool for functional genomics in C. elegans, whereas biochemical characteristics of RNAi hamper its use in mammalian cells. We here analyze kinetic and quantitative aspects of mammalian RNAi in different cell lines using novel lentiviral constructs with double H1-shRNA expression cassettes located in the U3 region of the LTR. Using enhanced green fluorescence protein (EGFP) as a target gene for RNAi and red fluorescence protein (RFP) as surrogate marker for intracellular siRNA expression, we show that long-term siRNA expression mediates stable RNAi. Furthermore, RNAi-induced gene silencing varies from minimal to complete loss-of-function phenotypes within homogenous and between different cell populations. Interestingly, the extent of gene silencing correlates to lentiviral integrations as well as siRNA expression levels in target cells. Finally, we demonstrate functional gene silencing of a cytokine receptor gene in normal CD34(+) hematopoietic progenitor cells. These data suggest that the use of suitable lentiviral constructs to quantify siRNA expression in living cells may allow analysis of loss-of-function phenotypes in a dose-dependent manner in a wide variety of target cells.