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.
Saayman
Sheena M
SM
Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School, Wits, Johannesburg, South Africa.
Arbuthnot
Patrick
P
Weinberg
Marc S
MS
D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D004258:DNA Polymerase III / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D012342:RNA, Small Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D012342:RNA, Small Nuclear; C405486:U6 small nuclear RNA; D004258:DNA Polymerase III