Abstract
RNA interference technology (RNAi) has been demonstrated to provide an effective tool in ecdysone receptor research. This chapter describes specific examples in three insect models. (1) Selective RNAi of the expression of the endogenous messenger RNA encoding EcR of Sf9 tissue culture cells and replace- ment with EcRs from other insects should permit cell-based screening for potential insecticides against a variety of pests. (2) The applicability of the technique of RNAi in Bombyx mori-derived Bm5 cells combined with the availability of a robust ecdysone reporter assay opens the possibility to carry out screens for the function of Bombyx genes in the primary ecdysone response pathway. (3) RNA interference (RNAi) works very well in the model insect, the red flour beetle, Tribolium cas- taneum. Methods for rearing beetles, synthesis of double-stranded RNA (dsRNA), injection of dsRNA RNA and evaluation of its efficacy by qRT-PCR analysis are presented. RNAi- mediated knock-down in the expression of genes such as EcR, USP, E75, HR3, HR4, HR39, HR38, SVP, FTZ-F1 and HR51 caused derailment of development from larvae to pupae and pupae to adult stages. These studies confirm the role of these nuclear receptors in 20E signal transduction. Keywords RNA interference dsRNA Bombyx mori Spodoptera frugiperda Tribolium castaneum insect cell lines Bm5 Sf9 EcR, usp HR3 E75 FTZ- F1 Broad ecdysone-responsive genes ecdysone regulatory pathway molting, moulting metamorphosis nuclear receptors transcription factors
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