Aalto Antti P, Sarin L Peter, van Dijk Alberdina A, Saarma Mart, Poranen Minna M, Arumäe Urmas, Bamford Dennis H
Institute of Biotechnology and Department of Biological and Environmental Sciences, University of Helsinki, Finland.
RNA. 2007 Mar;13(3):422-9. doi: 10.1261/rna.348307. Epub 2007 Jan 19.
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 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 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.
RNA干扰（RNAi）的发现彻底改变了生物学研究，并且在治疗方面具有巨大潜力。由于各种RNAi应用都需要小双链RNA（dsRNA），因此需要有经济高效的方法来大量生产高质量的dsRNA。我们提出了两种新型的、灵活的基于病毒的系统，用于高效生产dsRNA：（1）一种体外系统，利用T7 RNA聚合酶和噬菌体6的RNA依赖性RNA聚合酶（RdRP）的组合来生成长度实际上不受限制的dsRNA分子；（2）一种基于含有6聚合酶复合物的载体状态细菌细胞的体内RNA复制系统，以产生实际上无限制数量的长达4.0 kb的dsRNA。我们表明，源自这些系统产生的dsRNA的小干扰RNA（siRNA）池显著降低了HeLa细胞中报告基因（eGFP）的表达，并阻断了培养的交感神经元中内源性促凋亡BAX的表达及随后的细胞死亡。
