Uptake of dsRNA in cells to evaluate the efficacy of RNAi in insects

The midgut, the second largest organ in the insect body, is the entry site for food and water as well as for bacteria, viruses and toxins. This organ consists of a simple epithelium and forms the first barrier for uptake and translocation in the body hemocoel after oral intake. In this paper the use of primary cultures of midgut cells of the Colorado potato beetle is discussed. Upon dissection, mature cells with brush border membrane are incubated with fluorescent labeled dsRNA, and these data are compared to continuous insect cell lines. In addition, cell incubations were done with different inhibitors of endocytosis processes. In parallel, a gut-specific transcriptome analysis was done evaluating the presence of RNAi core products and uptake/transport proteins. The main objective of this project was to investigate the impact of the uptake endocytosis kinetics for dsRNA for a better understanding of the efficacy of RNAi in insects.