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The tTA System.

We recommend to test the system first in a transient assay, making cotransfections with e.g.

The ratio of the tTA producing plasmid vs. the response plasmids is critical for these transient experiments. An excess of pUHD15-1 over pUHC13-3 (up to 100-fold) is advisable. These conditions assure high intracellular concentrations of tTA and consequently high occupancies of the tet operator sites by tTA. At the same time, these plasmid ratios give a low background while maintaining high expression potential. In the presence of Tc, the background synthesis of reporter enzymes decreases proportionally when the amount of the response plasmid is lowered. This is in contrast to activated levels (i.e. in the absence of Tc) which are affected to a much lesser extent by the relative amount of the plasmid.

For Ca-phosphate transfections or lipofections, one transfection reaction should be split between two tissue culture dishes. Similarly, after electroporation the cells should be divided between two dishes. In either case, Dox-HCl should be added to one of the plates (2 ng/ml for initial experiments; this concentration may be lowered in subsequent experiments) (1, 6).

From our experience it is not necessary to preincubate cells with Dox-HCl prior to transfection since the uptake of the antibiotic is very fast. However, it cannot be ruled out that some cell lines may behave differently.

Since the transactivator has to be synthesized in order to initiate expression of the gene of interest, the time period required for the detection of the respective protein may be longer as compared to constitutive expression.Thus, the time period for maintaining expression may have to be adjusted accordingly.

The residual activity of the CMV minimal promoter sequence (-53 to +75) located between the tet operators and the gene of interest may vary to some extent in different cell lines. Transient expression experiments should be performed for each particular cell line in parallel with HeLa cells to examine the intrinsic activity in each cellular context. In our experience, cotransfection of HeLa cells with pUHC13-3 and pUHD15-1 yields regulation factors between 100 and 1000-fold in luciferase activities after 24 h +/- Dox-HCl. For the respective double stable cell lines see (1). In case tTA dependent regulation in the cell line of interest is not as good as in HeLa cells (due to elevated luciferase activity in the presence of Dox-HCl), switching to a different minimal promoter should be considered. In NIH 3T3 cells we observed a rather high basal activity of PhCMV*-1. This problem could be circumvented by using a Tk-based minimal promoter. Concommitant, however, with a lower basal activity (in presence of Dox-HCl), is in this case a lower activation of the promoter upon Dox-HCl withdrawal.

When examining the results of the transient expression experiments, it has to be kept in mind that higher regulation factors are usually found in double stable cell lines due to the reduction of background synthesis after chromosomal integration of the response unit. If background expression (i. e. expression in the presence of Dox-HCl) is too high, one might consider exchanging the minimal promoter sequence. Our plasmids are constructed such that this exchange can be readily achieved (see sequence print-outs).

The rtTA System.

rtTA-dependent gene expression can be demonstrated in double transient experiments using

in absence or presence of 1 mg/ml doxycycline.

It should be emphasized, that transient experiments with rtTA are less straight forward than with tTA.

We have observed major differences between rtTA and rtTAnls , whereby rtTAnls yielded a higher basal activity (absence of doxycycline). So far, we have not analyzed whether this is due to the elevated accumulation of the protein in the nucleus upon transient overexpression, or to a somehow altered affinity of the protein to its cognate binding sequence. It is clear, however, that this effect is not that pronounced after integration of the tet operator containing response units into the chromosome. Therefore, for transient experiments a broad titration of the regulator vs the responsive plasmids (we routineously use 1:1 down to .001:1) is initially recommended. These findings are based on only a very limited number of cell lines tested, since our own focus with the rtTA system is on transgenic animals. Here rtTA is superior to rtTAnls.

As with tTA the full potential of the rtTA regulation system can only be exploited in stably transfected cells.

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