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 wit h 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 pS G5 (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 th e synthesis of an apparently complete ecdysone receptor in a stable ce ll line produced in this way. Nuclear extracts from this line exhibit specific binding activity for the D. melanogaster hsp 27 ecdysone resp onse element in mobility shift analyses. Ecdysteroid induction of repo rter gene activity has been demonstrated in Chinese hamster ovary cell s both by transient transfection analysis and in stably transfected ce ll lines which constitutively produce the D. melanogaster ecdysone rec eptor.