Optimization of an siRNA-expression system with an improved hairpin and its significant suppressive effects in mammalian cells
Optimization of an siRNA-expression system with an improved hairpin and its significant suppressive effects in mammalian cells
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DOI：
10.1002/jgm.556
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发表时间：
2004-07-01
影响因子：
3.5
通讯作者：
Taira, K
中科院分区：
文献类型：
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作者：
Miyagishi, M;Sumimoto, H;Taira, K
背景RNA干扰(RNAi)是一种通过将与基因互补的双链RNA引入细胞来特异性沉默单个基因的表达的现象。当使用小干扰RNA (siRNA)时，可以在哺乳动物细胞中观察到这种现象，并且作为后基因组时代反向遗传学最强大的工具而受到关注。几个小组开发了基于载体的 siRNA 表达系统，可以在活细胞中诱导 RNAi。方法我们在此描述了各种 siRNA 表达系统的比较分析，其中我们检查了茎长度、环序列以及茎序列中突变和/或凸起的插入对沉默效果和载体稳定性的影响。结果作为比较分析的结果，我们确定了以下优化的 siRNA 表达系统：U6 启动子驱动的发夹型 dsRNA 21 nt 茎长，仅有义链有 3 至 4 个突变，以及源自 microRNA 的优化 9 nt 环序列。此外，我们证明具有四环素调节的U6启动子的siRNA表达系统可能具有控制细胞中RNAi的潜力，并且HIV载体介导的siRNA表达盒转移到细胞中导致在低至5的感染复数下有效沉默靶基因。结论突变的发夹siRNA及其遗传稳定的编码载体对于基因敲低实验非常有用，并且可以进一步有利于利用RNAi的基因治疗。版权所有 (C) 2004 John Wiley Sons Ltd。
Background RNA interference (RNAi) is a phenomenon in which expression of an individual gene can be specifically silenced by introducing a double-stranded RNA, one complementary to the gene, into cells. This phenomenon can be observed in mammalian cells when small interfering RNAs (siRNAs) are used, and is receiving attention as the most powerful tool for reverse genetics in the post genome era. Several groups have developed vector-based siRNA-expression systems that can induce RNAi in living cells.Methods We describe here a comparative analysis of various siRNA-expression systems, in which we examined the effects of stem length, loop sequence and insertion of mutation(s) and/or bulges in the stem sequence on silencing effects and on the stability of the vectors.Results As a result of the comparative analysis, we determined the following optimized siRNA-expression system: U6 promoter-driven hairpin-type dsRNA with 21-nt stem length, three to four mutations in the sense strand only, and the optimized 9-nt loop sequence, derived from microRNA. Moreover, we demonstrate that the siRNA-expression system with a tetracycline-regulated U6 promoter(s) could have the potential to control RNAi in cells, and that the HIV vector-mediated transfer of an siRNA-expression cassette into cells resulted in efficient silencing of a target gene at a multiplicity of infection as low as five.Conclusion The mutated hairpin siRNAs and their genetically stable coding vectors could be very useful for gene knockdown experiments, and could further benefit gene therapy using RNAi. Copyright (C) 2004 John Wiley Sons Ltd.