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The inducible reporter gene plasmids, pG5-luc and pGAL4-SEAP, were transfected into the Panc02+RHeoSwitch-3 and -4 cells and stable transfectants were isolated as they were into the MBT-2+RHeoSwitch cells.
Clones Panc02+RHeoSwitch-3+luc-3C1 AND -1B1 and Panc02+RHeoSwitch-4+luc-3A1 and -4B3 were used for further characterization.
In order to understand the induction kinetics of the inducible system, cell clone MBT-2+RHeoSwitch-2+luc-5B3 was treated with GS-E and cells were harvested at different times to determine the time course of gene induction.
These results show that the RHeoSwitch gene induction system gives consistent results independent of the cell type being used, the reporter gene used to assess the activity, as well as the chromosomal integration site of the DNA encoding the RHeoSwitch components (compare Panc02+RHeoSwitch-3 and -4 described above) or the integration site of the reporter gene DNA (compare multiple reporter clones from each of three RHeoSwitch clones described above).
The sensitivity of the gene expression system to varying doses of GS-E was determined by treating duplicate samples of clone MBT-2+RHeoSwitch-2+luc-5B3 with varying concentrations of GS-E for 24 or 48 hours and dual luciferase assays were performed on the cell lysates (Figure 3B).