Sensitive, rapid and simple method for evaluation of ecdysteroid agonist activity based on the mode of action of the hormone

Abstract

An efficient evaluation system of insect molting hormone activity at gene expression level has been developed. Reporter plasmid DNA was constructed by ligation of ecdysteroid responsive element containing Drosophila melanogaster hsp27 gene 5′ upstream region with the firefly luciferase gene. The reporter plasmid was transfected into Kc cell line by the electroporation method and cultured 24hr with or without ecdysteroids. Then cell lysates were made to measure luciferase activity. Maximal of more than 80-fold luciferase activity induction was observed in the presence of 2.0×10^-7M of 20-hydroxyecdysone or 2.0×10^-5M of α-ecdysone. Apparent induction was observed with 4.0×10^-9M of 20-hydroxyecdysone and from the high reproducibility, 4.8ng of the hormone was enough for the detection of the activity. Therefore this system should be the most sensitive method of the ecdysteroid agonist evaluation systems.