Controlling gene activities via the Tet regulatory systems:
- A Trouble Shooting Guide (Jan.1996) -
Table of Contents
Essential Components of the Tc-controlled Regulatory System
To Get Started
Establishing Cell Lines that Stably Express the tTA or rtTA Gene
Establishing Double Stable Cell Lines
Activation of Expression
Is the VP16 Domain Poisonous? - Concuding Remarks
1. 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 the PhCMV*-1-luciferase unit stably
integrated 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 experiments with
other promoters shows that it is a strong promoter exceeding e. g. the
activity of he 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 suitable when 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!
2. Essential Components of the Tc-controlled Regulatory System
The tTA-encoding vectors.
The basic plasmid (pUHD15-1)  encodes a CMV promoter/enhancer, the coding
sequence for tTA and a SV40 polyadenylation site. An alternative plasmid,
pUHG15-1  contains the %szlig;-globin intron/poly (A) site instead
of the SV40 poly (A) site. This may be particularly useful when tTA expression
is examined by RNA analysis. In our experience, the presence or absence
of an intron does not result in different levels of tTA synthesis.
We recommend the hCMV promoter/enhancer for tTA expression since it
has been used successfully in a number of cell lines. Depending on the
specific application, particularly when tissue-specific expression in transgenic
organisms is the goal, one has to consider more appropriate expression
signals. The optimal promoter/enhancer can only be defined in the context
of an experiment with respect to a particular cell line or organism.
We strongly recommend not to modify the tTA ORF! Several attempts (including
our own) e. g. insertion of nuclear localization sequences have resulted
in a hampered regulatory system.
The rtTA-encoding vectors.
Two versions of rtTA have been described (2). rtTA as encoded in pUHD17-1
or pUHG17-1 differs from tTA only by 4 amino acid exchanges, resulting
in the reverse phenotype. Beside the respective nucleotide exchanges pUHD17-1
is identical to pUHD15-1 (see above). For analytical purpose, the two plasmids
can be distinguished by an additional HindIII site in the rtTA coding region.
rtTA-nls as encoded by pUHD172-1neo contains an additional N-terminal nuclear
localization sequence (2). In addition, this plasmid contains a neomycin
The response plasmids such as pUHD10-3 (5) and its derivatives can be used
for the tTA as well as for the rtTA system. They have a tTA-dependent promoter
in front of a multiple cloning site or of a gene of interest. We strongly
recommend to examine tTA or rtTA function with a tTA-controlled reporter
unit. Our prefered reporter system is luciferase (pUHC13-3). If luciferase
is not a suitable reporter, pUHG16-3 (10) encoding tTA-controlled %szlig;-galactosidase
can be used.
The Effector Substance.
We now use preferentially doxycycline hydrochloride (Dox-HCl, which, like
tetracycline hydrochloride, is water soluble!) which functions with
tTA and rtTA. We use a freshly made stock solution of Dox-HCl for 2 weeks
(1 mg/ml; filter-sterilized and stored at 4oC in the dark).
Alternatively, stock solutions can be frozen in small aliquots at -20oC,
for long term storage. Dox-HCl in PBS forms a precipitate after a few days;
medium supplemented with Dox-HCl should therefore not be stored for prolonged
times. Tetracycline-HCl is only marginally functional in the rtTA system.
The most potent rtTA effector so far identified by us is doxycycline-HCl.
To feed doxycycline to transgenic animals, we routineously dissolve this
substance at a final concentration of 200 µg/ml in deionized water
(water bottle should be light protected). The drinking water which should
be renewed every 3 days contains 5 % succrose.
In our as well as in several other laboratories, it has been observed that
highly regulatable cell lines suddenly do not express the gene of interest
anymore when incubated in "Tc-free" medium. A 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 and we know today that commercial
serum preparations can shut off tTA responsive promoters. We suspect that
they contain Tc or one of its derivatives. We are in the process of identifying
such tetracyclines in some batches of serum. A good way to examine the
quality of sera in this respect is to grow and regulate the X1 cell line
described in our 1992 PNAS-paper. This cell line will soon be available
through the ECACC (European Collection of Cell Cultures, CAMR, Salisbury,
Wiltshire SP4 0JG, UK, Tel. +44-1980-612512, Fax +44-1980-611315).
3. To Get Started
The tTA System.
We recommend to test the system first in a transient assay, making cotransfections
The ratio of the tTA producing plasmid vs. the response plasmids is
critical for these transient experiments. An excess of pUHD15-1 over
pUHC13-3 (up to 100-fold) is advisable. These conditions assure high intracellular
concentrations of tTA and consequently high occupancies of the tet operator
sites by tTA. At the same time, these plasmid ratios give a low background
while maintaining high expression potential. In the presence of Tc, the
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 Tc) which are affected to a much lesser
extent by the relative amount of the plasmid.
pUHD15-1 (tTA-expression vector)
pUHC13-3 (luciferase-control vector) or pUHG16-3 (ß-gal-control vector)
(an unregulated internal standard may be included to examine the transfection
your gene of interest cloned into e.g. pUHD10-3.
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 (2 ng/ml for initial experiments;
this concentration may be lowered in subsequent experiments) (1, 6).
From our experience 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.
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.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. In our experience,
cotransfection of HeLa cells with pUHC13-3 and pUHD15-1 yields regulation
factors between 100 and 1000-fold in luciferase activities after 24 h +/-
Dox-HCl. For the respective double stable cell lines see (1). In case tTA
dependent regulation in the cell line of interest is not as good as in
HeLa cells (due to elevated luciferase activity in the presence of Dox-HCl),
switching to a different minimal promoter should be considered. In NIH
3T3 cells we observed a rather high basal activity of PhCMV*-1.
This problem could be circumvented by using a Tk-based minimal promoter.
Concommitant, however, with a lower basal activity (in presence of Dox-HCl),
is in this case a lower activation of the promoter upon Dox-HCl withdrawal.
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. Our plasmids are constructed
such that this exchange can be readily achieved (see sequence print-outs).
The rtTA System.
rtTA-dependent gene expression can be demonstrated in double transient
in absence or presence of 1 µg/ml doxycycline.
pUHG17-1 or pUHD172-1neo
one of the tTA/rtTA-dependent reporter units for your gene of choice cloned
e. g. into pUHD10-3 (see above),
It should be emphasized that transient experiments with rtTA are less
straight forward than with tTA.
We have observed major differences between rtTA and rtTAnls ,
whereby rtTAnls yielded a higher basal activity (absence of doxycycline).
So far, we have not analyzed whether this is due to the elevated accumulation
of the protein in the nucleus upon transient overexpression, or to a somehow
altered affinity of the protein to its cognate binding sequence. It is
clear, however, that this effect is not that pronounced after integration
of the tet operator containing response units into the chromosome. Therefore,
for transient experiments a broad titration of the regulator vs the responsive
plasmids (we routineously use 1:1 down to .001:1) is initially recommended.
These findings are based on only a very limited number of cell lines tested,
since our own focus with the rtTA system is on transgenic animals. Here
rtTA is superior to rtTAnls.
As with tTA the full potential of the rtTA regulation system can only
be exploited in stably transfected cells.
4. Establishing Cell Lines that Stably Express the tTA or rtTA Gene.
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 makes 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
The identification of rtTA-positive clones has, of course, to be carried
out in presence of 1 µg/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).
5. Establishing Double Stable Cell Lines.
Upon integration of a gene of interest into pUHD10-3, the resulting vector
should be transfered into the tTA or rtTA-positive cell lines by cotransfection
together with a second selection marker. Selection markers or other constructs
having 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. Therefore, 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
6. Activation of Expression
Induction by withdrawal or by addition of Dox-HCl may take some time to
be completed. This is not necessarily an intrinsic limitation of the regulatory
system itself, but rather primarily a function of the half-lives of the
mRNA and the protein under study: it will take some time before stably
expressed proteins accumulate to equilibrium levels.
In any case, suitably regulatable clones should be subcloned. This has
on occasion resulted in the identification of clones with even further
improved regulatory properties.
7. Is the VP16 Domain Poisonous? - Concluding Remarks
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
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
In conclusion, although there may 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 !
Gossen, M. and Bujard, H. (1992) Tight Control of Gene Expression in Mammalian
Cells by Tetracycline Responsive Promoters. Proc. Natl. Acad. Sci. USA
Gossen, M., Freundlieb, S., Bender, G., Müller, G., Hillen, W. and
Bujard, H. (1995) Transcriptional activation by tetracycline in mammalian
cells. Science 268, 1766-1769.
Gossen, M., Bonin, A. and Bujard, H. (1993) TIBS 18, 471-475.
Gossen, M., Bonin, A.L., Freundlieb, S. and Bujard, H. (1994) Inducible
gene expression systems for higher eukaryotic cells. Curr. Opin. Biotech.
Gossen, M. and Bujard, H. (1995) Efficiency of Tetracycline-controlled
Gene Expression is influenced by cell type: A commentary.. Biotechniques
19, No. 2.
Ackland-Berglund, C.E. and Leib, D.A. (1995) Efficiency of a tetracycline-controlled
gene expression is influenced by cell-type. Biotechniques 18, 196-200
Howe, J.R. et al (1995) The Responsiveness of a Tetracycline-sensitive
expression system differs in Different Cell Lines. J. Biol. Chem. 270,
Miller, K. and Rizzino, A. (1995) The Function of inducible Promotor Systems
in F9 Embryonal Carcinoma Cells. Exp. Cell Res. 218, 144-150
Shockett, P. et al (1995) A modified tetracycline-regulated system provides
autoregulatory inducible gene expression in cultured cells and transgenic
mice. Proc. Natl. Acad. Sci. 92, 6522-6526
Resnitzky, D., Gossen, M., Bujard, H. and Reed, S.I. (1994) Acceleration
of the G1/S phase transition by expression of cyclins D1 and E using an
inducible system. Mol.Cell.Biol. 14, 1669-1679.
Gossen, M. and Bujard, H. (1993) Anhydrotetracycline, a novel effector
for tetracycline controlled gene expression systems in higher eukaryotic
cells. Nucleic Acids Res. 21, 4411-4412.