Versatile Vectors for Ponasterone A- Inducible Control of Gene Expression in,,,Mammalian Cells ( resistant clones were screen...)

Versatile Vectors for Ponasterone A- Inducible Control of Gene Expression in,,,Mammalian Cells

resistant clones were screened for ponA-dependent induction of luciferase activity. Figure 5 shows the results for this double-stable cell line designated HSL-34. The results in Figure 5A show that both murA and ponA induce luciferase activity in HSL-34 cells to comparable levels over a 100-fold range of inducer concentration. To more accurately assess the sensitivity of the system, both ponA concentration and induction time were varied. As the results in Figure 5B indicate, a linear response was observed from 80 nM ponA (four fold: the lowest concentration tested) to 10 M.

To more directly assess the control of protein expression in this cell line, we performed a Western blot. Lysates from uninduced cells and cells induced with increasing amounts of ponA were fractionated by SDS-PAGE, blotted, and probed with a-luciferase polyclonal antisera (Figure 5C ). No significant detectable luciferase is expressed in the uninduced extract, whereas a linear increase in signal is observed from 300 nM to 5 M ponA. In a time-course experiment, a five-fold induction was achieved only 1 hour after adding 10 mM ponA, and a linear increase was observed for up to 20 hours, at which time an induction ratio of 1,030-fold was observed (Figure 5D ). We also observed these induction kinetics in transient assays (data not shown).

Fig.5

Taken together, these results indicate that, by varying the ponA concentration, precise control of gene dosage can be achieved with this system. Furthermore, comparatively brief induction periods can result in low to moderate levels of gene expression.

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( resistant clones were screen...)Versatile Vectors for Ponasterone A- Inducible Control of Gene Expression in,,,Mammalian Cells