The recent emergence of inducible expression systems for mammalian cells has greatly facilitated the in vivo analysis of gene function. The ecdysone-inducible expression system is particularly attractive because of ( i ) extremely low basal expression and high-level induced expression, ( ii ) the lack of pleiotropic effects caused by the inducer or activator, and ( iii ) the rapid penetrance and clearance of the inducer. Here, we describe an improved receptor expression vector. The required

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... sone receptor proteins (VgEcR and RXR) are co-expressed from a bicistronic cytomegalovirus (CMV) expression cassette in the vector pERV3. The CMV promoter in this vector can be readily replaced with a cell type-specific promoter of interest. Using the ecdysone analogs, muristerone A or ponasterone A, induction ratios of up to three orders of magnitude were attained in the transient transfection assays and in a cell line stably transformed with both pERV3 and an ecdysone-inducible reporter vector. Fine control of luciferase expression was achieved by varying both the induction time and inducer concentration. Here, we describe a set of cell lines stably transformed with the vector pERV3, in which the ecdysone receptors are expressed at optimal levels for the high-level induction of gene expression. Figure 4. PNA dose-responsiveness and kinetics of induction for the double stable cell line HSL-34. All of the results in panels A-C represent the average relative light unit (RLU) value for duplicate samples. Inductions were carried out for 20 h unless otherwise indicated.