Patent ID: 8637237

Claim:
A method for the identification of species-specific and stage-specific insecticides having the ability to potentiate ecdysteroid-activated ecdysone receptor (EcR)-mediated transcription in a species-specific manner comprising the steps of: (a) transfecting a first mammalian cell population with a first vector comprising a nucleotide sequence that encodes an Ultraspiracle (USP) polypeptide from a first species, a second vector comprising a DNA sequence that encodes an EcR isoform polypeptide, and a reporter gene operably linked to a hormone responsive element (HRE); (b) transfecting a second mammalian cell population with a vector comprising a nucleotide sequence that encodes a USP polypeptide from a second species, a second vector comprising a DNA sequence that encodes for an EcR isoform polypeptide, and a reporter gene operably linked to a hormone responsive element (HRE); (c) adding to individual aliquots of each of the mammalian cell populations of (a) and (b) either: (i) various doses of an ecdysteroid, (ii) various doses of a compound to be tested for its ability to potentiate ecdysteroid-activated EcR-mediated transcription, or (iii) various doses of a combination of the ecdysteroid and the compound to be tested for its ability to potentiate ecdysteroid-activated EcR-mediated transcription; (d) measuring whether there is an increase in EcR-mediated transcription in the cells for each of the aliquots in step (c) when the compound to be tested for its ability to potentiate ecdysteroid-activated EcR-mediated transcription is added with the ecdysteroid, as compared to when only the ecdysteroid is added, or when only the compound to be tested for its ability to potentiate ecdysteroid-activated EcR-mediated transcription is added; (e) assessing the ability of the compound to be tested for its ability to potentiate ecdysteroid-activated EcR-mediated transcription to increase the ability of the ecdysteroid to increase EcR-mediated transcription at sub-maximal doses of the ecdysteroid up to, but not greater than the maximal response evoked by the ecdysteroid, such that a compound that increases ecdysteroid-activated EcR-mediated transcription at sub-maximal doses of the ecdysteroid but does not increase the maximal response evoked by the ecdysteroid, and does not increase EcR-mediated transcription when added alone, comprises a potentiator compound; and (f) comparing the ability of the compound to be tested for its ability to potentiate ecdysteroid-activated EcR-mediated transcription to potentiate ecdysteroid-activated EcR-mediated transcription of the reporter gene in the first mammalian cell population of (a) and the second mammalian cell population of (b).