Numerous people have expressed concerns about the "poisonousness" of the VP16 domain. Just recall Paracelsus (1493 - 1541) who already pointed out that whether a compound may be a precious drug or a poison is often a question of concentration! It is common knowledge that many gene products, and in particular regulatory proteins such as transcription factors, function "physiologically" only when their intracellular concentration is limited to a defined window. Due to their high activation potential, VP16 fusion proteins like tTA/rtTA may require a lower concentration window than other transcription factors. However, random integration of the tTA/rtTA expression unit into cellular genomes and careful screening for the proper stable clones yield cell lines which fulfill exactly this requirement. For example, our HtTA-1 and X1 HeLa cell lines contain less than 10.000 tTA molecules per cell (probably around 6000) which, however, are sufficient to activate PhCMV*-1 more than 105 fold. Both cell lines grow in our lab without selection pressure since more than 5 years. Another about 15 cell lines established in our and other laboratories appear to behave analogously.
Similarly, we keep a number of tTA and rtTA mouse lines whose members live happily since generations, thanks to the proper concentration window of tTA/rtTA. Modifying the activating domain for lower "poisonousness" would most likely result in higher intracellular concentrations as compared e. g. to tTA. But for physiological function there would again be an upper limit. Thus, unless there are special reasons for changing the activating domain (e. g. to control specific interactions or to achieve tissue specificity, etc.), we do not see a need so far for abandoning the VP16 domain.
Is autoregulation of tTA (A. Bonin, M. Gossen and H. Bujard, unpublished; ref. 9) advantageous? As long as an autoregulatory system as described in ref. 9 is used, a rather complex situation is generated. Here, PhCMV*-1 - a very strong promoter when fully activated - controls tTA/rtTA synthesis. Upon induction, the transactivator may be quickly overexpressed to "poisonous" levels, which we feel lie > 10.000 molecules/cell. Thus, unless one likes to generate a "run-a-way" system which does not need to remain physiological, one has to limit the tTA/rtTA production via Dox-HCl. However, this may limit the expression of the gene of interest - also controlled by PhCMV*-1 - to concentrations not appropriate for the intended study. Therefore by autoregulating tTA by the same promoter which drives the gene of interest, the regulatory range of the latter is limited. This may be overcome by adjusting the individual strength of the two tTA-responsive promoters involved which, however, may turn out not to be as simple as it may appear at first glance.
In conclusion, although there may also be cell lines where stable expression of tTA/rtTA to a proper level is not tolerated (our experience does not support this view), in general there appears to be no need to control tTA/rtTA expression in cultured cell lines. Moreover, in transgenics the overwhelming number of approaches will require tissue specificity of tet control which we feel cannot be achieved readily by an autoregulatory system of the type described in ref. 9.
And here a notion of encouragement: In a time where clever combinations of commercially available kits and receipies appear to make experimental skills and an understanding of a system unnecessary, just keep in mind that the tet-system is not (yet?) a kit and that it is worthwhile to spend some thoughts on the basics - its fun and may be rewarding !