Wits-SAMRC Antiviral Gene Therapy Research Unit, Department of Molecular Medicine & Haematology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa.
Methods Mol Biol. 2020;2115:185-197. doi: 10.1007/978-1-0716-0290-4_11.
RNA interference (RNAi) is a promising tool for the treatment of chronic viral infection, such as that caused by the hepatitis B virus (HBV). RNAi activators, including expressed primary microRNA (pri-miRNA) mimics, can effectively silence viral gene expression and thereby inhibit viral replication. Here we describe a protocol for the design, generation and functional assessment of cassettes encoding effective single and multimeric pri-miRNA mimics. Artificial miRNAs targeting viral genes can be identified in silico and used to design corresponding pri-miRNA mimics. A two-step generation and TA cloning protocol can be used to produce single mimics, while the strategic use of restriction sites enables concatenation of mimics in a sub-cloning protocol. Basic gene silencing function of pri-miRNA mimics in cell culture can then be assessed using a dual luciferase assay and appropriate minimal targets. The methods described here for the generation of effective pri-miRNA mimics targeting HBV can be applied in the silencing of other viral or endogenous genes.
RNA 干扰 (RNAi) 是治疗慢性病毒感染的一种有前途的工具，例如乙型肝炎病毒 (HBV) 感染。RNAi 激活剂，包括表达的初级 microRNA (pri-miRNA) 模拟物，可以有效地沉默病毒基因表达，从而抑制病毒复制。本文描述了一种用于设计、生成和评估编码有效单聚体和多聚体 pri-miRNA 模拟物的盒的方案。可以在计算机上识别针对病毒基因的人工 miRNA，并用于设计相应的 pri-miRNA 模拟物。两步法生成和 TA 克隆方案可用于生成单模拟物，而限制酶位点的策略可用于在亚克隆方案中串联模拟物。然后可以使用双荧光素酶测定法和适当的最小靶标评估 pri-miRNA 模拟物在细胞培养中的基本基因沉默功能。本文描述的针对 HBV 的有效 pri-miRNA 模拟物的生成方法可用于沉默其他病毒或内源性基因。
