Logo ZMBH-Abteilung Prof. Dr. Hermann Bujard
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General Remarks

Leakiness, tightness and regulation factors.

Although we have discussed these issues in our various publications (ref 1 - 5), letters we receive as well as some recently published work (ref 6 - 9) demonstrate that they may have to be clarified once more. We define "leakiness" of a minimal promoter-tet operator construct such as PhCMV*-1 (1), as the intrinsic activity of such a sequence upon transfer into cells. When, for example pUHC13-3 encoding the luciferase gene under the control of PhCMV*-1 is transiently transfected into HeLa cells, the luciferase activity observed depends on (a) the intrinsic residual activity of PhCMV*-1, (b) the number of copies in the cell (which depends on the amount of DNA used for transfection). The intrinsic activity of the minimal promoter may vary in different cell lines, it may also change when additional sequence elements, which could function as enhancers, are introduced into the vector. Thus, to examine the suitability of a minimal promoter in a given vector and in a given cell line, transient experiments should be performed in which residual activities obtained under defined conditions are compared with those monitored in HeLa cells. Should the residual activities of the minimal promoter drastically exceed the values observed with pUHC 13-3 in HeLa cells, one might have to modify the vector or switch to a different minimal promoter sequence.

When a transcription unit controlled by a proper minimal promoter-tet operator sequence is integrated into the chromosome, the situation changes profoundly. After packaging into chromatin suppression, the residual activity of such promoters is drastically reduced. On the other hand, since such minimal promoters function also as enhancer traps, they may be activated by nearby enhancers. There may also be transcriptional read-through from outside promoters. Thus, in stable cell lines the so-called leakiness is primarily a function of a particular integration site.

This is demonstrated by the finding that cell lines like X1 (1) constitutively producing tTA and containing stably integrated the PhCMV*-1-luciferase unit show no measurable luciferase activity in presence of tetracycline (Tc). In absence of Tc, luciferase can be highly stimulated by tTA. Thus, it can be concluded that, by these criteria, PhCMV*-1 is not leaky in HeLa cells. The regulation factors observed in such cell lines can exceed values of 105. It has to be kept in mind, however, that such cell lines were identified after screening for low or no luciferase background in the non-induced state.

Quantitation of luciferase activity in our X1 HeLa cell line (1), indicates that in the uninduced state there are less than 7 molecules of the enzyme (detection limit) present in the cell. This shows that the system, if properly set up, is not only very tight but can also be highly active (induction factor > 105). We like to emphasize these facts again since several misleading reports were published recently (ref. 6,7,8) in which the regulation factors observed in transient experiments were compared to those described above and in ref. 1. Comparing the activity of the fully activated PhCMV*-1 in transient expression experimentswith other promoters shows that it is a strong promoter exceeding e. g. the activity of the hCMV promoter in HeLa cells and B cell lines (unpublished).Thus, the PhCMV*-1 is a tTa/rtTA-responsive promoter with a particularly broad range of regulation which is also suitablewhen high amounts of RNA are required as e. g. in anti- message approaches.

The tTA versus the rtTA system.

Although we are aware of experimental approaches where the use of the reverse tetracycline-controlled transactivator (rtTA) is advantageous, we do not share the views expressed in many letters we received recently. We do not feel that the rtTA system is "much better" than the tTA system, we also have no reason to assume that it is "much tighter" than the authentic (tTA) system. Even the "problem of having continuously tetracycline in the cell culture" is not really an issue. For example, using doxycycline at 1 ng/ml (which is about 1000 fold below cytotoxic concentrations) is sufficient to inactivate tTA. Without knowing, you may actually have used since years sera in your cell culture media which contain much higher tetracycline concentrations (see below)!

We would like to emphasize again that both systems are truly complementary and that the decision which one to use depends on the particular experimental strategy. To keep a gene switched off and to induce it rapidly at a given time, the rtTA system may be preferable. On the other hand, to keep a gene active and to turn it off occasionally, the tTA system may be better. Thus, don't throw out your tTA-producing cell lines or transgenics - only because molecular biologists like to induce gene activities by adding an effector substance following the paradigm of the E.coli lac operon!

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