Generating DNA Expression Cassettes Encoding Multimeric Artificial MicroRNA Precursors.

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.