Leadership In Controlled Gene Expression

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1. General Remarks

The following section is an update of the "troubleshooting guide" orginally published on Hermann Bujard`s web page. We will focus on the basic principles of the Tet regulation systems for 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 additional information on 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 such as tightness of control, expression levels and regulation factors, it is crucial to distinguish between two experimental situations ( (1) integrated state with stable insertion in the cell genome in tissue culture or (2) non-integrated state as in transient or episomal expression).  "Leakiness" of a minimal promoter-tet operator construct such as Ptet-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 Ptet-1 is transiently transfected into HeLa cells, the luciferase activity observed depends on (a) the intrinsic residual activity of Ptet-1 (b) the number of gene 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 activity of the minimal promoter drastically exceed the values observed with pUHC13-3 in HeLa cells, one may switch to Ptet-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.

Cell lines like X1 (Gossen and Bujard 1992 PNAS), which constitutively produce tTA and contain the Ptet-1-luciferase unit stably integrated show no measurable luciferase activity in the presence of tetracycline (Tc). In the absence of Tc, luciferase can be highly stimulated by a tTA. Thus, it can be concluded that, by these criteria, Ptet-1 is not leaky in HeLa cells. The induction 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 tightly regulated but can also be highly active (induction factor > 105). Comparing the activity of the fully activated Ptet-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 Ptet-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: 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. In 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 appears to be the more appropriate since its default state (i.e. absence of Dox) is inactive. In contrast, whenever an active gene is to be occasionally turned off, the tTA (Tet-Off) system is the system of choice.

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 for rtTA 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 the Ptet-1 promoter. These findings prompted mutagenesis studies 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 such as reduced background properties and particularly the higher sensitivity towards Dox (see below) when compared with the "original" 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, depending 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 provided evidence that tTA-mediated activation is fully shut off in less than 5 min after supplying Dox to cultured cells (Clement et al. 1999 RNA 5: 206). 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 the appearance of a phenotype based on the alteration of a gene's activity also depends critically on other parameters such as the halflife of the mRNA and the protein encoded by the gene of interest, despite the quick response of the Tet systems to their effector molecules. Thus, proteins with longer half-lives 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 more critical.

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4. Effector Substance

Doxycycline hydrochloride (Dox-HCl, which, like tetracycline hydrochloride, is water soluble) is the preferred effector substance, functioning 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 up to 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 binds to serum proteins thus serum-containing culture medium supplemented with Dox-HCl should not be stored for prolonged times.

It has been reported that highly regulatable cell lines suddenly do not express the gene of interest when incubated in tetracycline-free ("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 to either use commercially available tetracycline-free medium or to examine new batches of sera for the absence of tetracyclines by using the X1 or other existing, highly regulated cell lines.

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5. Setting up the System in Cell Culture

5.1. The tTA-dependent System.

It is recommended to test the system first in a transient assay format, for example co-transfection with:

  • pUHD15-1 or pUHD15-1neo (pTet-Off / tTA-expression vector) or pUHT61-1 (encoding tTA2-syn)
  • one of the Tet-responsive reporter units (e.g. pUHC13-3 p(TRE-luc)) and your gene of choice cloned e. g. into pUHD10-3 / pTRE (see above) or pTRE-Tight
  • an unregulated internal standard may be included to control transfection efficiency

The ratio of the tTA encoding plasmid relative to the response plasmids is critical for these transient experiments. An excess of pUHD15-1 or pUHT61-1 over pUHC13-3 or plasmids encoding the gene of interest is generally preferable. These conditions assure high intracellular concentrations of transactivator and consequently high occupancies of the tet operator sites. At the same time, these plasmid ratios generally result in low background transcription levels in the non-induced state 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 induced expression levels (i.e. in the absence of Dox) which are affected to a much lesser extent by the relative plasmid amount.

For Ca-phosphate transfections or lipofections, one transfection reaction should be split between two tissue culture dishes. Similarly, the cells should be divided between two dishes after electroporation. In either case, Dox-HCl should be added only 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 molecule.

Since the transactivator has to be synthesized 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 of each cellular context. Cotransfection of HeLa cells with pUHC13-3 (TRE-luc) and pUHD15-1 (Tet-Off) yields regulation factors between 102 and 103-fold in luciferase activities after 24 h +/- Dox-HCl. For results with the 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, higher regulation factors are usually detected in double stable cell lines due to the background reduction after chromosomal integration of the response unit. If background expression (i. e. expression in the presence of Dox-HCl) is too high, one may also consider exchanging the minimal promoter sequence.

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5.2. The rtTA-dependent System.

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

  • pUHG17-1 or pUHD172-1neo (pTet-On) or pUHrT62-1 encoding rtTA2-syn1
  • one of the Tet-responsive reporter units (e.g. pUHC13-3 p(TRE-luc)) and your gene of choice cloned e. g. into pUHD10-3 / pTRE (see above) or pTRE-Tight
  • an unregulated internal standard may be included to examine transfection efficiency

Of note, transient experiments with the improved Tet-On version highly efficient gene regulation can be monitored as readily as with the Tet-Off system. As with the tTA (Tet-Off) system, the full potential of the rtTA (Tet-On) / rtTA2 syn 1 regulation system can only be exploited in stably transfected cells.

The improved rtTA (rtTA2-syn1) is of particular interest as it confers the full induction range of the Tet 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.

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5.3. Establishing Cell Lines that Stably Express the transactivator gene.

It is recommended to establish the regulatory system in two steps: First, a stable cell line expressing the transactivator of choice should be constructed and characterized. In a second step, this line should be used for the transfer of the gene of interest. This approach will yield transactivator-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 transactivator-positive cell line allows the insertion of a variety of genes under control of a tet-responsive promoter. By contrast, after cotransfection of the regulator and the response plasmid, quantitative differences will occur due to different expression levels or integration loci of the transactivator construct. Thus, a direct 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. Hence, the enhancer of PhCMV driving the transactivator is brought into the proximity of the minimal promoter which can result in increased basal activity of the minimal promoter.

Two strategies were successfully applied to generate double stable cell lines: (i) cotransfection of the plasmid encoding the transactivator gene with a selection marker (like SV2neo) or (ii) 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. For some cell lines analysed, the first approach seems to result in cell lines with a better induction range. However, this observation stems from a limited number of experiments and is not yet 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 clones. In addition, the indirect assay by supertransfection will allow functional analysis of the clones rather than just demonstrating performed protein presence of the protein in the respective cells.

The identification of positive clones expressing the reverse transactivator (rtTA/Tet-On or rtTA2syn1) must, of course, be performed in the 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. Thus, it is prevented that the low intracellular concentration of the transactivator becomes limiting (to meet transfection requirements, unspecific DNA may be added)

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5.4. Establishing Double Stable Cell Lines.

Upon integration of the gene of interest into a tet responsive promoter, the resulting vector should be transferred into transactivator-positive cell lines by cotransfection together with a second selection marker. Selection markers or other constructs with proven or suspected enhancer activity should be avoided since the cotransfected DNAs will frequently cointegrate and the minimal promoter may serve as an enhancer trap. Enhancerless selection markers are recommended to obtain cells with a low basal expression level. This should, however, be a less critical concern when the goal is only a conditional overexpression.

An alternative to resistance markers are fluorescent markers (e.g. GFP) that may be cotransfected to permit sorting of transfected clones. Identified suitably regulatable clones´should be subcloned. This has on occasion resulted in the identification of clones with even greater ranges of induction.

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