TABLE 2.
Tetracycline-regulated expression of the wild-type CAT gene in the HeLa-tetOff cell line
| Vector(s) | Tc (1 μg/ml) | Avg CAT activity ± SD (U) | Fold regulation |
|---|---|---|---|
| pCMV-βgal (1 μg) | − | 12,434 ± 2,471 | 327 |
| pSVBpUC (1 μg) | + | 39 ± 5 | |
| pUtetO-CATwt (50 ng) | |||
| pUtetO-CATwt (5 ng) | − | 1,019 ± 7 | >1,018 |
| + | <1 ± 0.5 | ||
| pUtetO-CATwt (0.2 ng) | − | 219 ± 5 | ND |
| + | <1 ± 0.5 | ||
| None (mock) | − | <1 ± 0.5 |
HeLa-tetOff cells in six-well plates were cotransfected by using SuperFect in duplicate with three vectors, 1 μg of pCMV-βgal, 1 μg of pSVBpUC-sup2, and various amounts of pUtetO-CATwt as indicated. Where indicated, cells were incubated in the presence of 1 μg of tetracycline (Tc) per ml for the course of the transfection. CAT activity was determined by using various amounts of protein extracts that yielded activities within a linear range. One unit of CAT activity is defined as nanomoles of chloramphenicol acetylated by 10 μg of protein per hour; values are averages of duplicate transfection experiments. Fold regulation was calculated after normalization by subtracting the background CAT activity in mock-transfected cell extracts. ND, not determined.