Yu Jenn-Yah, DeRuiter Stacy L, Turner David L
Mental Health Research Institute, Program in Neuroscience, and Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109-0669, USA.
Proc Natl Acad Sci U S A. 2002 Apr 30;99(9):6047-52. doi: 10.1073/pnas.092143499. Epub 2002 Apr 23.
Duplexes of 21-nt RNAs, known as short-interfering RNAs (siRNAs), efficiently inhibit gene expression by RNA interference (RNAi) when introduced into mammalian cells. We show that siRNAs can be synthesized by in vitro transcription with T7 RNA polymerase, providing an economical alternative to chemical synthesis of siRNAs. By using this method, we show that short hairpin siRNAs can function like siRNA duplexes to inhibit gene expression in a sequence-specific manner. Further, we find that hairpin siRNAs or siRNAs expressed from an RNA polymerase III vector based on the mouse U6 RNA promoter can effectively inhibit gene expression in mammalian cells. U6-driven hairpin siRNAs dramatically reduced the expression of a neuron-specific beta-tubulin protein during the neuronal differentiation of mouse P19 cells, demonstrating that this approach should be useful for studies of differentiation and neurogenesis. We also observe that mismatches within hairpin siRNAs can increase the strand selectivity of a hairpin siRNA, which may reduce self-targeting of vectors expressing siRNAs. Use of hairpin siRNA expression vectors for RNAi should provide a rapid and versatile method for assessing gene function in mammalian cells, and may have applications in gene therapy.
21个核苷酸的RNA双链体，即小干扰RNA（siRNA），导入哺乳动物细胞后可通过RNA干扰（RNAi）有效抑制基因表达。我们发现，利用T7 RNA聚合酶进行体外转录可合成siRNA，这为化学合成siRNA提供了一种经济的替代方法。通过使用这种方法，我们证明短发夹siRNA能像siRNA双链体一样发挥作用，以序列特异性方式抑制基因表达。此外，我们发现基于小鼠U6 RNA启动子的RNA聚合酶III载体表达的发夹siRNA或siRNA可有效抑制哺乳动物细胞中的基因表达。U6驱动的发夹siRNA在小鼠P19细胞的神经元分化过程中显著降低了神经元特异性β-微管蛋白的表达，表明该方法对分化和神经发生的研究应具有实用性。我们还观察到，发夹siRNA中的错配可增加发夹siRNA的链选择性，这可能会减少表达siRNA的载体的自我靶向作用。使用发夹siRNA表达载体进行RNAi应为评估哺乳动物细胞中的基因功能提供一种快速且通用的方法，并且可能在基因治疗中具有应用价值。
