Defining the Role of Subolesin in Tick Cell Culture by Use of RNA Interference

Abstract

Development of tick vaccines provides new opportunities for control of tick infestations and tick-borne diseases. Recently, the tick-protective protein, subolesin, was identified in a cell line derived from Ixodes scapularis by expression library immunization and a mouse model of tick infestations. While subolesin was conserved among ixodid tick species, the biological function of this gene is unknown. Subolesin expression in ticks was silenced by RNA interference (RNAi) to provide information on the gene's function, and silencing of subolesin profoundly impacted tick survival, feeding, and reproduction. In this research we used RNAi in the IDE8 tick cell line to further study the role of subolesin in development of cultured tick cells. The cells were incubated with subolesin double-stranded (ds)RNA and cell growth was monitored. Incorporation of dsRNA by tick cells was monitored with Cy3-labeled dsRNA. After 72 h cells were harvested for cell counts, morphology, and for confirmation of gene silencing by reverse transcriptase-PCR. While the expression of subolesin in treated cells was reduced 80 +/- 9% by RNAi as compared with mock-treated cells, cell growth did not appear to be affected over the 72-h period. This is the first report of the use of RNAi in tick cell culture. RNAi is a powerful tool for studying tick gene function and will likely contribute to our understanding of the role that tick genes play in cell development and infection with pathogens.