Use of Bombyx mori U6 promoter for inducing gene-silencing in silkworm cells

Abstract

Double-stranded (ds) RNA is a useful tool for sequence-specific gene silencing, now known as RNA interfer ence (RNAi), in a variety of organisms. Studies on pol III promoter-based expression systems have been well documented showing them to be an effective tool for inducing RNAi in mammalian cells and Drosophila Schneider 2 (S2) cells, but not in silkworm cells. In this study, we developed a Bombyx mori U6 promoter (U6p)based short hairpin RNA (shRNA) expression system for inducing RNAi in silkworm cells. First, a transient reporter assay showed that the transfection of plasmids containing U6p-driven shRNA-expression units (U6-shRNA units) caused a decrease in the expression of the target gene in BmN cells with the efficacy dependent on the length of the hairpin stem region. Subsequently, we demonstrated the ability of the U6-shRNA unit to confer resistance to Bombyx mori nucleopolyhedrovirus (BmNPV)-resistance on BmN cells; i.e., the viral titer in the culture medium of BmN cells transfected with U6-shRNA (with the sequence of an essential viral gene)-expression plasmids was decreased to 30% of the control at 48 hr post-infection. These results suggested that the U6shRNA expression system was a useful tool for RNAi in silkworm cells.