Functional transfer of an elementary ecdysone gene regulatory system to mammalian cells: Transient transfections and stable cell lines

A 3.1 Kb fragment of a Drosophila melanogaster ecdysone receptor (EcR) cDNA (splice product, EcR B1) comprising the 2.6 Kb coding region with 218 base pairs of 5' and 258 base pairs of 3'-untranslated sequence, was cloned into the mammalian expression vectors pH beta APr-1 and pSG5 (which place EcR under the control of a human beta-actin and a SV40 early promoter, respectively). Chinese hamster ovary cells have been stably transfected with the beta-actin promoter construct. Antiserum, prepared against an EcR-fusion protein has been used to demonstrate the synthesis of an apparently complete ecdysone receptor in a stable cell line produced in this way. Nuclear extracts from this line exhibit specific binding activity for the D. melanogaster hsp 27 ecdysone response element in mobility shift analyses.Ecdysteroid induction of reporter gene activity has been demonstrated in Chinese hamster ovary cells both by transient transfection analysis and in stably transfected cell lines which constitutively produce the D. melanogaster ecdysone receptor.