Large-scale production of dsRNA and siRNA pools for RNA interference utilizing bacteriophage φ6 RNA-dependent RNA polymerase
Large-scale production of dsRNA and siRNA pools for RNA interference utilizing bacteriophage φ6 RNA-dependent RNA polymerase
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DOI：
10.1261/rna.348307
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发表时间：
2007-03-01
期刊：
影响因子：
4.5
通讯作者：
Bamford, Dennis H.
中科院分区：
文献类型：
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作者：
Aalto, Antti P.;Sarin, L. Peter;Bamford, Dennis H.
RNA干扰（RNAi）的发现彻底改变了生物学研究，并具有巨大的治疗潜力。由于各种RNAi应用需要小的双链RNA（dsRNA），因此需要用于产生大量高质量dsRNA的具有成本效益的方法。我们提出了两种新的，灵活的基于病毒的系统，用于有效生产dsRNA：（1）利用噬菌体φ 6的T7 RNA聚合酶和RNA依赖性RNA聚合酶（RdRP）的组合以产生实际上无限长度的dsRNA分子的体外系统，以及（2）一种基于含有phi 6聚合酶复合物的载体状态细菌细胞的体内RNA复制系统，最大为4.0 kb的dsRNA。我们表明，池的小干扰RNA（siRNA）来自这些系统产生的dsRNA显着降低了HeLa细胞中的转基因（eGFP）的表达，并阻止内源性促凋亡BAX表达和随后的细胞死亡在培养的交感神经元。
The discovery of RNA interference (RNAi) has revolutionized biological research and has a huge potential for therapy. Since small double-stranded RNAs (dsRNAs) are required for various RNAi applications, there is a need for cost-effective methods for producing large quantities of high-quality dsRNA. We present two novel, flexible virus-based systems for the efficient production of dsRNA: (1) an in vitro system utilizing the combination of T7 RNA polymerase and RNA-dependent RNA polymerase (RdRP) of bacteriophage phi 6 to generate dsRNA molecules of practically unlimited length, and (2) an in vivo RNA replication system based on carrier state bacterial cells containing the phi 6 polymerase complex to produce virtually unlimited amounts of dsRNA of up to 4.0 kb. We show that pools of small interfering RNAs (siRNAs) derived from dsRNA produced by these systems significantly decreased the expression of a transgene (eGFP) in HeLa cells and blocked endogenous pro-apoptotic BAX expression and subsequent cell death in cultured sympathetic neurons.