The Repressor configuration. A. Transient transfection: Five micrograms of reporter (pAdCMV5-CuOs-LacZ or pAdCMV5-CuOg-LacZ) were transfected alone (lanes 2 and 5 respectively) or co-transfected with 250 ng repressor construct (pAdCMV5-CymR) (lanes 3, 4 and 6, 7) in 293 cells. Three micrograms of pcDNA-SEAP was included as an internal control of transfection efficiency. Transfections were carried out in the presence (4, 7) and absence (lanes 1, 2, 3, 5 and 6) of 200 μg/ml cumate. β-galactosidase (β-gal) activity was measured 48 h post-transfection using a colorimetric assay. Reporter activity was normalized to SEAP activity in the culture medium. pAdCMV5-LacZ was used as a reference for promoter strength (lane l). The figure represents data from 3 independent experiments. B. Screening 293-CymR clones: Clones of 293 stably expressing CymR were infected with AdCMV5-CuOs-LacZ (MOI 10) in the presence and absence of 200 μg/ml cumate. β-galactosidase activity was measured 48 h post-infection using a colorimetric assay in cell extracts. Clone 21, marked with an arrow was retained for further experimentation. C. Stable expression of reporter: 293-CymR-CMV5-CuO-GFP cells were cultured in the presence and absence of 200 μg/ml of cumate for 48 h. The figure shows flurescent and phase contrast images.