Boudreau Ryan L, Davidson Beverly L
Department of Internal Medicine, University of Iowa, Iowa City, Iowa, USA.
Methods Enzymol. 2012;507:275-96. doi: 10.1016/B978-0-12-386509-0.00014-4.
RNA interference (RNAi) is a natural process of gene silencing mediated by small RNAs. Shortly after the discovery of the RNAi mechanism, scientists devised various methods of delivering small interfering RNAs (siRNAs) capable of co-opting the endogenous RNAi machinery and suppressing target gene expression based on sequence complementarity. RNAi has since become a powerful tool to study gene function and is being investigated as a potential therapeutic approach to treat a vast array of human diseases (e.g., cancer, viral infections, and dominant genetic disorders). Among the available RNAi vectors are hairpin-based expression platforms (short-hairpin RNAs and artificial microRNAs) designed to mimic endogenously expressed inhibitory RNAs. These RNAi vectors are capable of achieving long-term potent gene silencing in vitro and in vivo. Here, we describe methods to design and generate these hairpin-based vectors and briefly review considerations for downstream applications.
RNA干扰（RNAi）是由小RNA介导的一种天然基因沉默过程。在RNAi机制被发现后不久，科学家们就设计出了各种递送小干扰RNA（siRNA）的方法，这些小干扰RNA能够借助内源性RNAi机制，基于序列互补性来抑制靶基因表达。自那以后，RNAi已成为研究基因功能的强大工具，并且正在作为一种潜在的治疗方法来研究，用于治疗一系列人类疾病（如癌症、病毒感染和显性遗传病）。现有的RNAi载体中，有基于发夹结构的表达平台（短发夹RNA和人工微小RNA），其设计目的是模拟内源性表达的抑制性RNA。这些RNAi载体能够在体外和体内实现长期有效的基因沉默。在此，我们描述了设计和生成这些基于发夹结构载体的方法，并简要回顾了下游应用的注意事项。
