A Simple and Robust Vector-Based shRNA Expression System Used for RNA Interference

BackgroundRNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful genetic tool for conducting functional studies. Previously, vector-based shRNA-expression strategies capable of inducing RNAi in viable cells have been developed, however, these vector systems have some disadvantages, either because they were error-prone or cost prohibitive.
ResultsIn this report we described the development of a simple, robust shRNA expression system utilizing 1 long oligonucleotide or 2 short oligonucleotides for half the cost of conventional shRNA construction methods and with a>95% cloning success rate. The shRNA loop sequence and stem structure were also compared and carefully selected for better RNAi efficiency. Furthermore, an easier strategy was developed based on isocaudomers which permit rapid combination of the most efficient promoter-shRNA cassettes. Finally, using this method, the conservative target sites for hepatitis B virus (HBV) knockdown were systemically screened and HBV antigen expression shown to be successfully suppressed in the presence of connected multiple shRNAs both in vitro and in vivo.
ConclusionThis novel design describes an inexpensive and effective way to clone and express single or multiple shRNAs from the same vector with the capacity for potent and effective silencing of target genes.

背景：由小干扰RNA（small interfering RNAs, siRNAs）或短发夹RNA（short hairpin RNAs, shRNAs）介导的RNA干扰（RNA interference, RNAi）现已成为功能研究领域的强力遗传工具。此前，已有可在活细胞中诱导RNAi的基于载体的shRNA表达策略被开发，但这类载体系统存在一些缺陷：要么易出错，要么成本过高难以承担。
结果：本研究报道了一种简便且高效的shRNA表达系统，仅需1条长寡核苷酸或2条短寡核苷酸即可完成组装，其构建成本仅为传统shRNA构建方法的一半，克隆成功率超过95%。研究中还对shRNA的环序列与茎部结构进行了比对与精心筛选，以提升RNAi的沉默效率。此外，基于同尾酶（isocaudomers）开发了更为便捷的策略，可快速组合最优的启动子-shRNA表达盒。最终，利用该方法系统筛选了乙型肝炎病毒（hepatitis B virus, HBV）的保守靶位点，并证实联合表达多条shRNAs可在体内外成功抑制HBV抗原的表达。
结论：本新型设计提供了一种低成本且高效的方法，可从同一载体中克隆并表达单条或多条shRNAs，实现对靶基因的强效、有效沉默。