RNA interference in uninfected and baculovirus infected lepidopteran cells.

摘要

RNA interference (RNAi) is a cellular mechanism capable of suppressing gene expression in many organisms. RNAi refers to the induction of gene silencing machinery by the introduction of double-stranded RNA (dsRNA) homologous to the gene to be suppressed. Few such studies have been done on the lepidopteran cells, Spodoptera frugiperda (Sf9). In this work Sf9 cells have been used to express the marker gene for the enhanced green fluorescent protein (EGFP) either transiently, by transfecting cells with plasmids containing this gene, or via infection with recombinant baculoviruses expressing EGFP under the polyhedrin promoter.; The EGFP gene has been delivered into the Sf9 cells either as a single gene, or fused to a scorpion gene (LqqIT2), or after the LqqIT2 gene but separated by an internal ribosomal entry site (IRES) sequence (bicistronic RNA). The inducers in this gene silencing system were EGFP dsRNA, a small interference RNA (siRNA) consisting of 22 base pairs from the EGFP coding region, and the EGFP antisense RNA. Also a transformed Sf9 cell line constitutively expressing two copies of the EGFP gene in opposite orientations, to produce an inverted repeat that would form a dsRNA, was established to suppress EGFP expression. The transient expression of the EGFP gene was totally suppressed when the dsRNA and siRNA were the inducers of the RNA interference mechanism. Sf9 cells infected with recombinant baculoviruses expressing EGFP were capable of suppressing the expression of EGFP with either of the inducers but less efficiently than the transient expression. The stably transformed cells also were found to partially suppress gene expression. Knocking down genes in Sf9 cells transiently and via recombinant baculovirus infection can help in increasing the yield of recombinant baculoviruses that could be potentially used as biopesticides. This silencing system can also help understand the RNAi machinery in the Sf9 cells and can be applicable to mammalian cells in studying mammalian viruses.