Jian Rui, Peng Tao, Deng Shaoli, Jiang Jing, Hu Fuquan, An Jing, Cheng Xiaoxing
Laboratory of Infection Immunity, Department of Microbiology, Third Military Medical University, Chongqing 400038, PR China.
Eur J Cell Biol. 2006 May;85(5):433-40. doi: 10.1016/j.ejcb.2005.10.009. Epub 2005 Dec 7.
RNA interference (RNAi) is a powerful tool for functional genetic studies in model organisms and mammalian cells. To facilitate rapid construction of gene knockdown constructs and RNAi libraries for known genes of mammalian cells, a new and simple strategy to produce small interfering RNA (siRNA) expression vectors with two opposing polymerase III promoters was developed. The design involved a one-step PCR amplification and single cloning procedure to construct a dual promoter siRNA expression vector. The forward primer is identical for all PCR reactions, only a single reverse primer that contains the siRNA targeting sequence has to be synthesized in the construction of each individual vector. This single primer design is cost-effective and it reduces the risk of sequence errors during synthesis of long oligos. Sense and antisense strands of siRNA duplexes were transcribed from the same template and this eliminated the need to synthesize long hairpin-forming oligonucleotides. Our study demonstrated that this vector design could mediate potent inhibition of expression of both exogenous and endogenous genes in mammalian cells.
RNA干扰（RNAi）是用于模式生物和哺乳动物细胞功能基因研究的强大工具。为便于快速构建针对哺乳动物细胞已知基因的基因敲低构建体和RNAi文库，开发了一种新的简单策略，用于生产带有两个反向聚合酶III启动子的小干扰RNA（siRNA）表达载体。该设计涉及一步PCR扩增和单克隆程序，以构建双启动子siRNA表达载体。所有PCR反应的正向引物相同，在构建每个单独载体时，只需合成一个包含siRNA靶向序列的反向引物。这种单引物设计具有成本效益，并降低了长寡核苷酸合成过程中序列错误的风险。siRNA双链体的正义链和反义链从同一模板转录而来，这消除了合成长发夹形成寡核苷酸的必要性。我们的研究表明，这种载体设计可介导对哺乳动物细胞中外源和内源基因表达的有效抑制。
