Figure 5

A. Screening of 293-CR5-LacZ clones. G418-resistant clones were infected with AdCMV-cTA in the presence and absence of 200 μg/ml cumate. Forty-eight hours later, β-Gal activity was measured. The figure shows the subclone # 13 displays high-level expression and the best ON/OFF ratio, almost 40-fold. B. Screening adenoviral library of cTA mutants. 293CR5-LacZ cells were infected with the adenoviral library of CymR mutants and overlayed with agarose supplemented with Blue-gal. Plaques formed by the infection of adenovirus expressing the wild-type cTA are β-gal positive in the absence of cumate, and β-gal negative in the presence of 200 μg/ml cumate. C. The plaque indicated by an arrow is a candidate for the reverse phenotype, since high β-gal expression is evident in the presence of 200 μg/ml cumate.