Lentiviral-Mediated RNA Interference

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Abstract
RNA interference (RNAi) is a form of posttranscriptional gene silencing mediated by short double-stranded RNA, known as small interfering RNA (siRNA). These siRNAs are capable of binding to a specific mRNA sequence and causing its degradation. The recent demonstration of a plasmid vector that directs siRNA synthesis in mammalian cells prompted us to examine the ability of lentiviral vectors to encode siRNA as a means of providing long-term gene silencing in mammalian cells. The RNA-polymerase III dependent promoter (H1-RNA promoter) was inserted in the lentiviral genome to drive the expression of a small hairpin RNA (shRNA) against enhanced green fluorescent protein (EGFP). This construct successfully silenced EGFP expression in two stable cell lines expressing this protein, as analyzed by fluorescence microscopy, flow cytometry, and Western blotting. The silencing, which is dose dependent, occurs as early as 72 hr postinfection and persists for at least 25 days postinfection. The ability of lentiviruses encoding siRNA to silence genes specifically makes it possible to take full advantage of the possibilities offered by the lentiviral vector and provides a powerful tool for gene therapy and gene function studies.