Cheng Tsung-Lin, Chang Wen-Tsan
National Cheng Kung University Medical College, Tainan, Taiwan.
Methods Mol Biol. 2007;408:223-41. doi: 10.1007/978-1-59745-547-3_13.
RNA interference (RNAi) is an evolutionarily conserved mechanism of posttranscriptional gene silencing induced by introducing the double-stranded RNAs (dsRNAs) into cells. Recent progress in RNAi-based gene-silencing techniques has revolutionarily advanced in studies of the functional genomics and molecular therapeutics. Among the widely used dsRNAs including exogenously synthetic and endogenously expressed small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs), the shRNAs are more efficient than siRNAs on the induction of gene silencing and currently have evolved as an extremely powerful and the most popular gene silencing reagent. The DNA vector-based shRNA-expression systems provide not only a simple and effective way in inhibiting gene activities in either inheritable or inducible manner, but also a cost-effective tool in constructing the expression vectors. To fully explore the DNA vector-based shRNA-expression systems in RNAi-mediated gene-silencing techniques, four distinct RNA polymerase III (Pol III)-controlled type III promoter-based expression vectors are constructed including pHsH1, pHsU6, pMmH1, and pMmU6, which contain either the RNase P RNA H1 (H1) or small nuclear RNA U6 (U6) promoter from human and mouse. Moreover, to improve the constructing and screening efficiency for the shRNA-expression recombinant clones, these four DNA vectors are further reconstructed by inserting a stuffer of puromycin resistance gene (PuroR) between restriction enzyme ClaI and HindIII sites, which makes the preparation of vectors easy and simple for cloning the shRNA-expression sequences. Because of the ease, speed, and cost efficiency, these four improved DNA vector-based shRNA-expression vectors provide a simple, convenient, and efficient gene-silencing system for analyzing specific gene functions in mammalian cells. Herein, the simple and practical procedures for the construction of DNA vector-based expression vectors, potential and rational design rules for the selection of effective RNAi-targeting sequences, efficient and cost-effective cloning strategies for the construction of shRNA-expression cassettes, and effective and functional activity assays for the evaluation of expressed shRNAs are described.
RNA干扰（RNAi）是一种进化上保守的转录后基因沉默机制，通过将双链RNA（dsRNA）导入细胞来诱导。基于RNAi的基因沉默技术的最新进展在功能基因组学和分子治疗学研究中取得了革命性的进展。在广泛使用的dsRNA中，包括外源性合成和内源性表达的小干扰RNA（siRNA）和短发夹RNA（shRNA），shRNA在诱导基因沉默方面比siRNA更有效，目前已发展成为一种极其强大且最受欢迎的基因沉默试剂。基于DNA载体的shRNA表达系统不仅提供了一种以可遗传或可诱导方式抑制基因活性的简单有效方法，也是构建表达载体的一种经济高效的工具。为了在RNAi介导的基因沉默技术中充分探索基于DNA载体的shRNA表达系统，构建了四种不同的基于RNA聚合酶III（Pol III）控制的III型启动子的表达载体，包括pHsH1、pHsU6、pMmH1和pMmU6，它们分别包含来自人和小鼠的核糖核酸酶P RNA H1（H1）或小核RNA U6（U6）启动子。此外，为了提高shRNA表达重组克隆的构建和筛选效率，通过在限制性内切酶ClaI和HindIII位点之间插入嘌呤霉素抗性基因（PuroR）填充片段，对这四种DNA载体进行了进一步改造，这使得载体的制备对于克隆shRNA表达序列变得简单易行。由于其简便性、速度和成本效益，这四种改进的基于DNA载体的shRNA表达载体为分析哺乳动物细胞中的特定基因功能提供了一个简单、方便且高效的基因沉默系统。本文描述了基于DNA载体的表达载体构建的简单实用程序、有效RNAi靶向序列选择的潜在合理设计规则、shRNA表达盒构建的高效经济克隆策略以及用于评估表达的shRNA的有效功能活性测定方法。
