TABLE 3.

Comparison of the modified (tTAnls) and unmodified (tTA) transactivator for activation of CAT expression in COS-1 cells^a

Vector transfected with pUtetO-CATwt	Tc (2 μg/ml)	Avg CAT activity ± SD (U)	Fold regulation
pUHD15-1 (pU-CMV-tTA)	—	3,823 ± 113	48
	+	80 ± 13
pU-CMV-tTAnls	—	55,944 ± 6,394	76
	+	739 ± 53
pCMV-βgal	—	<1 ± 0.5
None (mock)	—	<1 ± 0.5

^aCOS-1 cell cultures in 60-mm-diameter dishes were cotransfected with 2.5 μg of pUtetO-CATwt and 2.5 μg of either pUHD15-1 or pU-CMV-tTAnls, using the DEAE-dextran method. As a control, the same amount of pCMV-βgal was used with pUtetO-CATwt. Where indicated, cells were incubated in the presence of 2 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.