Scherr Michaela, Battmer Karin, Ganser Arnold, Eder Matthias
Zentrum Innere Medizin, Abteilung Hämatologie und Onkologie, Medizinische Hochschule Hannover, Carl-Neuberg Strasse 1, D-30623 Hannover, Germany.
Cell Cycle. 2003 May-Jun;2(3):251-7.
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 homogeneous 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.
RNA干扰（RNAi）介导由双链RNA引发的序列特异性转录后基因沉默。RNAi是秀丽隐杆线虫功能基因组学的有效工具，而RNAi的生化特性阻碍了其在哺乳动物细胞中的应用。我们在此使用新型慢病毒构建体分析不同细胞系中哺乳动物RNAi的动力学和定量方面，该构建体在LTR的U3区域含有双H1-shRNA表达盒。使用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表达的替代标志物，我们表明长期siRNA表达介导稳定的RNAi。此外，RNAi诱导的基因沉默在同质细胞群体内以及不同细胞群体之间从最小到完全功能丧失表型有所不同。有趣的是，基因沉默的程度与慢病毒整合以及靶细胞中的siRNA表达水平相关。最后，我们证明了细胞因子受体基因在正常CD34 +造血祖细胞中的功能性基因沉默。这些数据表明，使用合适的慢病毒构建体来量化活细胞中的siRNA表达可能允许以剂量依赖方式分析多种靶细胞中的功能丧失表型。
