1. General Remarks

The following section is an update and replacement of the ‘troubleshooting guide’ on Hermann Bujard’s webpage which we will continue as a service to the scientific community. We will focus here on the basic principles of the Tet regulation systems in tissue culture. Other biological systems in particular transgenic mice will be incorporated in the ‘Applications’ section. Detailed technical instructions for tissue culture and transgenic mice experiments can soon be found in the ‘Protocols’ section. An introduction to the basic components as well as improved versions of the system can be found elsewhere on this website.

For an extended discussion on the basic principles underlying the Tet regulation systems please refer to Baron and Bujard 2000 Meth.Enzymol. 327: 401-421 which is available as a free download.

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2. Tightness of Control and Regulation Factors

When considering properties of the Tet system like tightness of control, expression levels and regulation factors, it is crucial to distinguish between experimental situations where the systems are stably inserted in the genome of cells in culture and situations where they are in a non-integrated state as in transient expression experiments or when contained in episomes. Here “leakiness” of a minimal promoter-tet operator construct such as P_tet-1 is defined as the intrinsic activity of such a sequence upon transfer into cells. When, for example pUHC13-3 encoding the luciferase gene under control of P_tet-1 is transiently transfected into HeLa cells, the luciferase activity observed depends on (a) the intrinsic residual activity of P_tet-1 (b) the number of copies in the cell (which in turn 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. In case residual activities of the minimal promoter drastically exceed the values observed with pUHC13-3 in HeLa cells one may switch to P_tet-14 or alternatively one may have to modify the vector.

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, the residual activity of such promoters is drastically reduced. On the other hand, since minimal promoters function also as enhancer traps, they may be activated by nearby enhancers, furthermore there may also be transcriptional read-through from outside promoters. Thus, in stable cell lines the so-called leakiness is primarily a function of the particular integration site.

This is demonstrated by the finding that cell lines like X1 (Gossen and Bujard 1992 PNAS) constitutively producing tTA and containing the P_tet-1-luciferase unit stably integrated show no measurable luciferase activity in presence of tetracycline (Tc). In the absence of Tc, luciferase can be highly stimulated by tTA. Thus, it can be concluded that, by these criteria, P_tet-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 the X1 HeLa cell line (Gossen and Bujard 1992 PNAS) indicates that in the uninduced state less than 7 molecules of the enzyme (detection limit) are 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). Comparing the activity of the fully activated P_tet-1 in transient expression experiments with other promoters shows that it is a strong promoter exceeding e. g. the activity of the hCMV promoter in several cell lines analysed.Thus, the P_tet-1 is a Tet-responsive promoter with a particularly high dynamic range of regulation.

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3. The tTA versus the rtTA system and Kinetics of Induction

The availability of the tTA (Tet-Off) and rtTA (Tet-On) system raises the question under which conditions one should prefer the one over the other. At the level of cell culture, both systems are nearly equivalent: both permit the regulation of gene activity over several orders of magnitude and the kinetics of induction are fast. Nevertheless, if a gene should be kept inactive most of the time and turned on only occasionally, the rtTA (Tet-On) system seems more appropriate as it is inactive in its default state (i.e. absence of Dox). In contrast whenever an active gene is to be occasionally turned off, the tTA (Tet-Off) system is preferable.

The quantitative comparison of tTA (Tet-Off) and rtTA (Tet-On) revealed that the two transactivators are not fully equivalent. While HeLa tTA (Tet-Off) cell lines with a regulatory range of > 10⁵ and no detectable background activity could readily be obtained (Gossen and Bujard 1992 PNAS) analogously generated rtTA (Tet-On) based cell lines such as HR5-CL11 (Gossen et al Science 1995) generally exhibited a low but distinct basal activity in the absence of Dox. Nevertheless the regulation factors in properly screened clones can also exceed 10³-fold.

Further analysis of rtTA (Tet-On) revealed that it has a low residual affinity to tetO in the absence of Dox leading to elevated background activity of P_tet-1. These findings prompted mutagenesis of tTA (Tet-Off) DNA yielding several new rtTA mutants ( Urlinger et al. 2000). Some of the improved reverse trans­activators like rtTA2-syn1 have remarkable properties that make them fully complementary to tTA (Tet-Off). The advantages of rtTA2-syn1 like improved background properties and particularly the higher sensitivity towards Dox (see below) when compared with rtTA (Tet-On) are of particular interest when using the system in transgenic animals.

The induction of the tTA/rtTA (Tet-Off/Tet-On) systems in cell culture is fast since it depends largely on the diffusion constant of the antibiotic. Cells are rapidly equilibrated with Dox, but the antibiotic can also efficiently be washed out of a culture provided it is not used at excessive concentrations. A study designed to determine the halflife of short lived intron RNA ( Clement et al. 1999 RNA 5: 206) provided proof that tTA-mediated activation is fully shut off in less than 5 min after supplying Dox to cultured cells. The same kinetics can safely be assumed for the activation of the rtTA (Tet-On) system in cell culture.

It is important to keep in mind that despite the quick response of the Tet systems towards their effectors supplied from outside the appearance of a phenotype based on the alteration of a gene's activity depends decisively on other parameters such as the halflife of the mRNA and the protein encoded by the gene of interest. Thus proteins with longer halflife will require more time than short lived proteins to reach steady state levels as they continue to accumulate.

In transgenic organisms saturation/depletion with doxycycline of different compartments of an organism follows complex pathways which will largely determine the kinetics of activation or inactivation of a gene. There, the choice between the two regulatory systems will be of greater relevance.

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4. Effector Substance

Doxycycline hydrochloride (Dox-HCl, which, like tetracycline hydrochloride, is water soluble) so far is the preferred effector substance which functions efficiently with tTA (Tet-Off) rtTA (Tet-On) and rtTA2-syn1. A freshly made stock solution of Dox-HCl (1 mg/ml in H₂O; filter-sterilized and stored at 4 °C in the dark) can be used for 2 weeks. Alternatively, stock solutions can be frozen in small aliquots at -20 °C, for long term storage. Dox-HCl in PBS forms a precipitate after a few days furthermore the antibiotic is also bound by serum proteins thus medium supplemented with Dox-HCl should not be stored for prolonged times.

It has been noticed that highly regulatable cell lines suddenly do not express the gene of interest anymore when incubated in "Tc-free" medium. Thorough analysis of several of these incidents revealed that in each case a change in the batch of calf serum or fetal calf serum had occurred. Subsequently it was shown that different serum preparations contain Tc or one of its derivatives that can shut off Tet responsive promoters. It is therefore important either to use commercially available tetracycline-free medium or to examine new batches of sera for the absence of tetracyclines by using the X1 or other highly regulated cell lines.

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The ratio of the tTA encoding plasmid vs. the response plasmids is critical for these transient experiments. An excess of pUHD15-1 or pUHT61-1 over pUHC13-3 or plasmid encoding gene of interest is generally advisable. These conditions assure high intracellular concentrations of transactivator and consequently high occupancies of the tet operator sites. At the same time, these plasmid ratios give a low background while maintaining high expression potential. In the presence of Dox 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 Dox) 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.

Generally 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 and require preincubation with the effector.

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 e.g. in stably transfected cells. 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. Cotransfection of HeLa cells with pUHC13-3 (TRE-luc) and pUHD15-1 (Tet-Off) yields regulation factors between 100 and 1000-fold in luciferase activities after 24 h +/- Dox-HCl. For the respective double stable cell lines see (Gossen and Bujard 1992 PNAS). In case tTA (Tet-Off) -dependent regulation in the cell line of interest is not as good as in HeLa cells alternative minimal promoters may be used as outlined above. 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.

The improved rtTA (rtTA2-syn1) is of particular interest as it confers full induction of the system at lower effector concentrations promising enhanced regulation in transgenic animals even in organs like the brain which so far were partially refractory to Tet regulation.

Two strategies were successfully applied to generate double stable cell lines: cotransfection of the plasmid encoding the transactivator gene with a selection marker (like SV2neo) or integration of the respective resistance cassette into the transactivator encoding vectors. In the latter case, the percentage of G418 resistant clones showing the expected transactivator mediated phenotype was definitively higher. However at least for some cell lines analysed the first approach seems to give higher-regulatable cell lines. However this observation stems from a limited number of experiments and is not well understood.

Once resistant clones are isolated they should be examined for transactivator expression via transient supertransfection with pTRE luc / pUHC13-3 +/- Dox (see above). This has proven to be the most reliable method for detection of the transactivator gene which generally is low and makes direct detection by band shift assays or immunoblotting in most cases difficult and unsuitable for screening of 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 positive clones expressing the reverse transactivator (rtTA/Tet-On or rtTA2syn1) has, of course, to be carried out in presence of Dox.

Should the transactivator-positive cell lines be used for transient experiments, involving tet-responsive promoters, it is important to use low amounts of the respective Tet-responsive expression plasmid for transfection. In this way, it is prevented that the low intracellular concentration of the transactivator becomes limiting (to meet transfection requirements, unspecific DNA may be added)

An alternative to resistance markers are fluorescent markers (e.g. GFP) that may be cotransfected and allow sorting of transfected clones.Upon identification of suitably regulatable clones these should be subcloned. This has on occasion resulted in the identification of clones with even further improved regulatory properties.