Fig. 2

Regulation of luciferase activity in NIH3T3 cells infected with vectors representing the“two virus”strategy

NIH3T3 cells were first infected with retroviral vectors for constitutive expression of the transactivators tTA, rtTA, or mock incubated with BOSC 23 supernatant alone, as indicated in the“1st vector”column. After splitting into multiple dishes, the three different NIH3T3 populations were superinfected with different vectors, as indicated in the“2nd vector”column. The next day the cultures were split into replica plates and gene expression was kept in the off state (solid bar) or turned on (shaded bar) through the addition of doxycycline in the case of rtTA expressing cells or removal of tetracycline in the case of tTA expressing cells. Luciferase activities of cell extracts were determined 48 hours after induction. Enzymatic activities are expressed in relative light units (rlu) and were normalized for the amount of total protein of the samples and the copy number of the luciferase gene in the different cell populations, which was determined by Southern blot analysis. Relative induction levels were calculated by dividing the enzyme activity of the on state through that of the off state from replica plates of the same culture, and are shown in the“relative induction”column.