We recommend to establish the regulatory system in two steps: a stable cell line expressing tTA or rtTA should be constructed and characterized first; in a second step, this line should be used for the transfer of the gene of interest. This approach will yield tTA- and rtTA-positive cell lines that can serve as a defined genetic background. These lines will then allow the direct comparison of different clones containing a subsequently introduced gene of interest. Moreover, a well defined tTA or rtTA positive cell line allows the insertion of a variety of genes under control of a tTA-responsive promoter. By contrast, after cotransfection of the regulator and the response plasmid, quantitative differences will also be due to different expression levels or integration loci of the tTA or rtTA construct. Thus, a strict comparison of the different clones is not possible. Moreover, during cotransfection, the expression unit of the transactivator may integrate at the same chromosomal locus (as it is frequently observed in cotransfection experiments). In this way, the enhancer of PhCMV driving tTA or rtTA is brought into proximity of the minimal promoter which can result in increased basal activity of the minimal promoter.
We have successfully used two strategies: cotransfection of pUHD15-1 or pUHD17-1 with a selection marker (generally SV2neo) or integration of the respective resistance cassette into the tTA or rtTA encoding vectors. In the latter case, the percentage of G418 resistant clones showing the expected tTA+ or rtTA phenotype is definitively higher. Still we prefer the first approach since it seems to give higher-regulatable cell lines. However this is an observation from a limited number of experiments and we have no good explanation for the difference.
Once resistant clones are isolated they should be examined for tTA or rtTA expression via transient supertransfection with pUHC13-3 or pUHG16-3 +/- Tc (see above). So far, we observed only limited expression of the tTA or rtTA gene after stable transfection of mammalian cells with pUHD15-1 or pUHD17-1. The low amount of transactivator in those cells make its direct detection by band shift assays or immunoblotting in most cases difficult and not suitable for screening many clones. In addition the indirect assay by supertransfection will provide a result on the functionality of the clones rather than just demonstrating the presence of the protein in the respective cells.
The identification of rtTA-positive clones has, of course, to be carried out in presence of 1 mg/ml of Dox.
Should the transactivator-positive cell lines be used for transient experiments, whereby the gene of interest is controlled by a tTA- or rtTA-responsive promoter, it is important to use low amounts of the respective expression plasmid for transfection. In this way, it is prevented that the low intracellular concentration of tTA or rtTA becomes limiting (to meet transfection requirements, unspecific DNA may be added).