Regulating rcTA expression. A. Schematic representation of the strategy. The expression of rcTA is controlled by the CMV5-CuO promoter. In the absence of cumate, CymR binding to CuO blocks the synthesis of rcTA. In the absence of rcTA, GFP expression from CR5-GFP is not stimulated. Leaky expression of rcTA is not sufficient to activate transcription from the CR5 promoter. Upon addition of 50 μg/ml cumate, CymR binding to CuO is abrogated and therefore rcTA is synthesized. Cumate binding to rcTA activates its DNA binding activity thereby turning on reporter gene expression. B. Screening of 293-rcTA and C. 293CymR-rcTA clones. The clones were infected with lenti-CR5-GFP in the presence and absence of 50 μg/ml cumate and 48 h later GFP fluorescence was measured by flow cytometry. D. Stable expression of reporter CR5-SEAP-IRES-GFP in 293-rcTA and 293CymR-rcTA: 293cTA, 293-rcTA and 293CymR-rcTA transduced with lenti-CR5-SEAP-IRES-GFP were cultured in the presence and absence of 50 μg/ml cumate for 48 h. SEAP activity was measured in the cell culture medium.