TABLE 5.
Quantitative regulation of in vivo suppression in COS-1 cells by tetracycline
| Vector transfected with pRSVCATam27 and pSVBpUC-sup2 | Tc (μg/ml) | CAT activity (U) | Normalized CAT activ- ity (U) | Fold regu- lation |
|---|---|---|---|---|
| pUHD10-3 + pCMV-βgal | 0 | 1 | ||
| pU-CMV-tTAnls | 0 | 1 | ||
| pUtetO-16GlnRS + pCMV-βgal | 0 | 3 | ||
| pUtetO-16GlnRS + pU-CMV-tTAnls | 0 | 37 | 34 | |
| pUtetO-16GlnRS + pU-CMV-tTAnls | 0.2 | 11 | 8 | 4 |
| pUtetO-16GlnRS + pU-CMV-tTAnls | 1.0 | 3 | 0 | >34 |
| pUtetO-16GlnRS + pU-CMV-tTAnls | 5.0 | 3 | 0 | >34 |
| None (mock) | 0 | 1 |
COS-1 cell cultures in 60-mm-diameter dishes were cotransfected with four vectors, 2.5 μg of pRSVCATam27, 1.25 μg of pSVBpUC-sup2, 2.5 μg of pUtetO-16GlnRS, and 2.5 μg of pU-CMV-tTAnls, by the DEAE-dextran method. For control experiments, pUtetO-16GlnRS and pU-CMV-tTAnls were replaced with pUHD10-3 and pCMV-βgal, respectively, where indicated. Cells were incubated with various amounts of tetracycline (Tc) between 0 and 5 μg/ml in medium. 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. Fold regulation was calculated after normalization by subtracting the background CAT activity (3 U) in cell extracts where pU-CMV-tTAnls is replaced by pCMV-βgal. Values represent averages of results from at least three independent transfection experiments.