Effective Pol III-Expressed Long Hairpin RNAs Targeted to Multiple Unique Sites of HIV-1

The RNA interference (RNAi) pathway has in recent years been exploited for the development of novel antiviral therapies. The emergence of viral escape mutants, however, is a major impediment to the use of RNAi effectors to treat highly mutable viruses such as HIV-1. A combinatorial approach is therefore required for long-term inhibition of gene expression. RNA Pol III-driven long hairpin RNA (lhRNA) duplexes can be cleaved several times by Dicer, yielding multiple functional siRNAs from a single construct. Here we describe a method for the generation of ectopically expressed U6-lhRNAs encoding three separate siRNA sequences targeting unique sites in HIV-1. This methodological overview explains some crucial aspects of lhRNA design and cloning as well as facile experiments to determine their efficacy in cell culture.