BACKGROUND
the tet system is the most widely used inducible gene expression technology in eukaryotes, for both, in vivo and in vitro applications.
based on its original design  <cit> , now frequently referred to as „tet-off" system, it experienced its first major modification by the introduction of the „tet-on" system  <cit> .
both systems respond in opposite ways to the presence of tetracyclines , by either inactivating  or activating gene expression .
the difference is based on amino acid exchanges in the synthetic transcription factors employed, tta and rtta respectively, reversing the response of the dna binding domain to the presence of the allosteric effector dox.
initially both systems relied on the same composite tet-responsive promoter, ptet- <dig>  <cit> .
subsequent attempts to broaden the utility and performance of the tet system focused on transactivator manipulation: nuclear localization sequences were introduced  <cit> , codon usage was optimized  <cit> , potential splice sites were removed  <cit>  and activation domains have been exchanged  <cit> .
the most significant advances, however, came from genetic approaches to identify improved versions of tet-on type transactivators.
these experiments aimed to improve the dynamic range, by either a reduction of residual dna binding  in the non-induced state or enhancement of activation in the induced state.
in this context, the most notable transactivator alleles were identified in yeast  <cit>  and, through enforced retroviral evolution, in human cells  <cit> .
while these approaches focused on improving the dynamic range of the tet system via modified transactivators, it appeared interesting to explore, whether the tet system could also be improved by manipulation of the cis-acting tet-responsive promoters.
the initially introduced ptet- <dig>  <cit>  is based on a minimal promoter fragment derived from the cmv immediate early promoter.
various alternatives were introduced over the years, like those further truncating the cmv minimal promoter of ptet- <dig>  <cit> , constructs based on the hsv tk promoter  <cit> , the mmtv long-terminal repeat promoter  <cit>  and the hiv- <dig> long-terminal repeat promoter  <cit> .
several of these ptet variants had their advantages under the specific conditions tested, with e.g.
a "second-generation tetracycline regulatable promoter"  <cit>  showing up to  <dig> orders of magnitude in inducible gene regulation experiments using the tet-off regulation principle.
however, with the exception of ptet- <dig> , which was derived from ptet- <dig> by empirical modifications and displayed a substantially reduced background expression, none of these promoter constructs is widely used.
here we present our results with a series of novel ptet variants.
systematically introduced alterations were generated to minimize background expression while maintaining high levels of induced expression.
to place these experiments in a proper context, it is worthwhile to initially distinguish between the different sources of such background expression.
especially in a comparative analysis of transient transfection and stable transduction experiments as presented here, their individual contribution can be readily addressed.
considering the various sources of background expression is an essential requirement when expression systems based on the transactivation paradigm are engineered and is not restricted to the tet system.
in principle, background expression can be triggered by residual binding of transactivators to their cognate binding sites.
this would be the case when expression, driven by a ptet promoter, is higher in a tet transactivator-positive cell line in the off-state as compared to an otherwise isogenic tet transactivator-negative cell line.
secondly, background expression due to e.g.
enhancers or other sequence elements able to act at long-range on ptet is to be expected and has indeed been frequently observed.
such effects can be avoided by targeted insertion of the ptet driven transcription unit into an appropriate genomic locus  <cit>  or suppressed by the use of insulator elements  <cit> .
thirdly, background expression is also influenced by the accessibility of the ptet, which is determined by epigenetic parameters such as dna methylation and integration into chromatin structures.
lastly, background expression might be intrinsic for a given ptet due to hidden binding sites for transcription factor.
reduction of this intrinsic background was the goal of the current study.
for this purpose, we designed novel ptet promoter variants with low background activities and highly induced expression levels by stepwise modification of ptet- <dig>  already known functional cis elements of the promoter where exchanged, others expected to optimize its performance were introduced and those suspected to impede performance where deleted.
this strategy proved successful when the new ptet promoter variants where quantitatively characterized in a plasmid vs. retrovirus backbone and examined in different cell lines.
our results revealed that several novel ptet promoters, and in particular, ptet-t <dig>  are superior to ptet- <dig> and ptet- <dig> with regard to low background levels in the off-state, while retaining a high activation potential.
RESULTS
experimental strategy
the tet system with its key elements ptet and the tetracycline controlled transcriptional activators  is outlined in figure 1a.
ptet- <dig> is composed of a cmv derived minimal promoter linked downstream to an array of seven tet operators.
the focus of this work was the further optimization of ptet- <dig> via reduction of background expression while maintaining or even improving the levels of induced expression.
such combined effects would result in an improved range of transcription control.
for comparison ptet- <dig> and ptet- <dig> were included in the study.
as outlined in figure 1b, these two promoters differ in the spacing between tet operators.
the center-to-center distance of neighboring operators is  <dig> nt for ptet- <dig> versus  <dig> nt for ptet- <dig>  moreover, their respective cmv minimal promoter moieties differ in length.
the t-series of ptet promoters introduced here all contain a tet operator heptamer with  <dig> nt spacing .
in contrast to ptet- <dig> where the spacer sequences between the operators are identical we used randomized spacer sequences , thus minimizing the symmetry within the operator region.
this accounts for the sensitivity of viral vectors to palindromic sequences due to the strand displacement mechanism  <cit> , which may lead to gradual loss of operators during reverse transcription  <cit> .
replacing the operator heptamer of ptet- <dig> with this newly designed operator array resulted in ptet-t <dig>
subsequent modifications of the minimal promoter moiety of the ptet promoters followed a rational design strategy, stepwise altering known functional sequence elements within the cmv minimal promoter as used in ptet-t <dig>  introducing point mutations to ptet-t <dig> led to consensus sequences of the tata-box and the tfiib binding site  in ptet-t <dig>  the bre upstream the tata-box could possibly enhance transcriptional initiation upon induction with acidic domain-type transactivator vp <dig>  <cit> , while reducing background transcription of promoters in the non-induced state  <cit> .
deletion of the cmv 5'-utr led initially to ptet-t <dig> with truncated downstream promoter element .
ptet-t <dig>  exhibiting a rudimentary 5'-utr, contained only the cmv-initiator element  directly followed by the start codon.
in order to restore an average length 5'-utr, we decided to introduce an unrelated eukaryotic sequence from a plant rna virus  fused 3' to the inr sequence of ptet-t <dig>  this sequence contains only few cis elements known to be important for promoter regulation in the animal kingdom .
the transcription factor binding sites found, for rfx- <dig> and ap- <dig>  were eliminated by point mutations, resulting in ptet-t <dig>  further modifications of the tymv 5'-utr led to ptet-t <dig> and ptet-t <dig>  as discussed in more detail below, these constructs are devoid of potential splice signals  and hairpin structures  <cit>  that could confer cap-independent translation initiation.
we were primarily interested in functional properties of the newly designed ptet promoters in heterogeneous, non-clonal cell populations.
we therefore evaluated these promoters by transient transfection of plasmid dna, as well as γ-retroviral transduction resulting in stable cell pools.
initial characterization of the new promoters in a plasmid backbone
the new ptet promoters as well as the control constructs , ptet- <dig> and ptet-14) were all integrated in an identical manner into the plasmid puhc-131- <dig>  <cit>  containing firefly luciferase as the reporter gene.
transfection into tet transactivator-positive hela-em <dig> cells  <cit>  was performed in the presence and absence of dox.
the results as shown in figure  <dig> demonstrate that all the new ptet promoters were highly inducible.
ptet-t <dig> displayed a 5-fold higher induced expression level than ptet- <dig>  although both contain the identical cmv minimal promoter.
ptet-t <dig> also displayed a 6-fold reduction of non-induced background expression resulting in a regulation factor of at least 13000-fold vs. 400-fold for the original ptet- <dig>  this improvement is solely based on the new operator heptamer introduced into otherwise identical constructs.
the expression characteristics of ptet-t <dig> were similar to that of ptet-t <dig>  differing only by the introduction of a consensus bre in the t <dig> construct.
modifications of the 5'-utr  led to slightly reduced expression in the induced state when compared to ptet-t <dig>  still, even the weakest of these ptet promoters, ptet-t <dig>  displayed a slightly higher induced expression level than the original ptet- <dig>
in line with our experimental objective, the more interesting outcome of the stepwise alteration of the minimal promoter when modifying the 5'-utr was the further reduction of background expression in the off-state, already obvious from shortening the cmv leader sequence in ptet-t <dig> and ptet-t <dig> .
introduction of the tymv 5'-utr and its variants  reduced background expression even further.
especially ptet-t <dig> displayed significantly lower background activity, more than 100-fold reduced compared to ptet- <dig> and about 10-fold compared to ptet- <dig>  this resulted in regulation factors of about 50000-fold in these transient transfection assays .
it should be noted here that for several of the ptet promoters the luciferase values measured by us were very close to instrument background.
compared to our established protocols we increased the amount of transfected plasmid dna in order to obtain readings that could be reliably quantified .
performance of the new regulatory units after retroviral transduction
the newly designed ptet promoters were integrated into the es.
<dig> γ-retroviral vector  containing lmg* as the reporter gene .
lmg* is a dual reporter gene providing gfp as well as luciferase activity .
this allowed us to determine differences in unregulated background expression with exceptional sensitivity in luciferase assays without sacrificing the option of single cell resolution via gfp measurements.
infections of htta- <dig> cells  were performed at low moi  under induced conditions .
this ensured that the majority of the transduced cells contained a single integrated copy of the response unit  <cit> .
the results shown in figure 3c were similar but not identical to those of the transfection experiments .
all new ptet promoters displayed high expression levels in the induced state, slightly higher than ptet- <dig>
a direct comparison of reporter activities in absolute terms is not possible between the different experiments, as we had to switch from luciferase activity normalized to a second co-transfected reporter  to luciferase activity normalized to the protein content of cellular extracts .
however, the pattern of background expression levels in the off-state of the system were similar between the different ptet promoters when comparing the results shown in figure  <dig> with those of figure  <dig>  as in case of the plasmid transfections the ptet-t <dig> transduced cell populations displayed the lowest background expression after chromosomal integration.
the only notable difference resulted from the introduction of the bre , which had a much more pronounced effect on background expression upon chromosomal integration.
comparison of the doxycycline dose response of ptet- <dig> and ptet-t6
next, we asked whether the altered ptet promoter architecture would result in dose response changes of the regulatory system towards dox.
these parameters were previously established for ptet- <dig> in hela cells.
hela-em <dig> cells were transduced by es.1-ptet-1p or es.1-ptet-t6p retroviral sin-vectors.
purified populations were incubated in the presence of the indicated dox concentration for  <dig> hours, harvested and analyzed for egfp fluorescence and luciferase actvity .
the gfp data confirm that ptet-t <dig> can reach a slightly improved expression level when compared to ptet- <dig>  since in the non-induced state, the mean fluorescence of both cell populations is too close to the background level of hela-em <dig> cells no meaningful conclusion  can be drawn.
taking advantage of the luciferase activity of the lmg* reporter, simultaneous luciferase measurements revealed that background expression in the off-state of ptet-t <dig> is 15-fold below that of ptet- <dig>  in line with the previous analysis .
the combination of reduced background and slightly enhanced induced expression level resulted in an about 30-fold increased regulation range.
the maximum induction for both promoters had been reached at  <dig> ng dox/ml; further increase of the effector concentration was without effect.
note that the steep increase of gene expression between  <dig> and  <dig> ng dox/ml is similar for both ptet promoters, indicating an identical synergistic response of both promoters to increased occupancy of the operators by tet-transactivators.
the facs analysis at single cell resolution  indicates that all cells responded to induction, but not homogenously.
upon partial induction  the expression level of the individual cells differed widely.
it has to be emphasized that the cell population analysis was genetically heterogeneous with respect to insertion of the reporter unit.
again, the overall characterization of the two ptet promoters analyzed revealed no major differences in the dose response parameters.
fine-tuning of transgene expression
the results obtained with the new ptet promoters, especially ptet-t <dig>  led us to routinely employ these promoters in ongoing research projects in our laboratories.
the following results were derived from a project aiming at the conditional expression of erbb <dig> in breast cancer cell lines.
a tet transactivator-positive mda-mb <dig> ro cell line expressing rtta-s <dig>  <cit>  has been previously established .
transduction of this clone was performed with a retroviral sin-vector, es.1-t6p , in which the erbb <dig>  open reading frame was under control of ptet-t <dig>  as the erbb <dig> protein is located on the cell surface, it is well suited for detection and selection of the transduced population by facs.
the results of the cell-based analysis  of the purified population showed, that  erbb <dig> expression was tightly regulated in these experiments with no recognizable background expression in the off-state,  despite looking at non-clonal pools expression could be adjusted to intermediate levels by titrating dox, and  cells could be maintained at a chosen expression level over a prolonged period of time.
this time course analysis was terminated on day  <dig>  interestingly, prolonged full induction  led to the disappearance of erbb <dig> cells, showing highest expression levels, as can be seen from the asymmetric steep drop of the right shoulder of the expression profile peak.
thus, the expression level appears to reflect the maximum long-term expression level of erbb <dig> tolerated by those cells.
the only other recognizable difference in the expression profiles was a slight „compression" of intermediate expression at  <dig> ng/ml dox, as the cell-to-cell variability in transgene expression was apparently reduced after prolonged growth under these conditions.
given the non-clonal origin of this cell population, individual cells behaved remarkably similar.
still, for certain applications, like analyzing pharmacological responses to varying densities of cell surface receptors, the homogeneity under intermediate expression conditions may not be sufficient for reliable results.
thus, we examined whether respective homogeneous cell populations, derived from cell clones, would improve the situation.
the example shown in figure 5b, lower panel, demonstrates that this is indeed the case.
further suppression of background expression could already be observed in the vast majority of cells, but the precision of partial induction was markedly improved by clonal isolation of cells harboring ptet-t <dig>
discussion
our goal was to improve the dynamic range of tetracycline regulated transcription in such a way that tight, virtually background-free non-induced expression can routinely be obtained when working with non-clonal cell populations.
the tet system in its current configuration has proven its capacity to achieve such background free expression, as widely documented in the literature.
however, most of these studies relied on clonal analysis of transgenic cells, thereby eliminating cell clones with less favorable properties, an often laborious process which at times might even be impractical, for example in the genetic engineering of primary cells.
by using previously established and well-characterized transactivator-positive cell lines we restricted our efforts solely to cis elements of tet-controlled transcription units, the tet-responsive promoters.
we investigated if gene expression regulated by a rationally designed series of new ptet promoters would be superior over the widely used original ptet- <dig> promoter and its derivative ptet- <dig> , particular by further reducing background expression of tet-regulated transcription units while maintaining fully-induced expression levels.
we conclude that the reduction of the ptet background expression reported here is exclusively due to the low intrinsic background of these new tet-responsive promoters.
their side-by-side analysis  in different established tet transactivator-positive cell lines eliminated the possibility that effects like transactivator abundance or differences in tta-vs. rtta-mediated transcription control would influence our conclusions.
moreover, the tet operator sequences themselves are identical in all new ptet promoters used and any potential residual transactivator affinity to the operators would not have any influence on the outcome of our study.
lastly, we excluded a systematic distortion of our results by the possible occurence of integration site-specific or epigenetic effects through parallel analysis of all ptet promoters in transient transfection assays.
the initial transition from ptet- <dig> to ptet-t <dig> resulted in a more than 5-fold lower background expression in transient transfection assays.
this effect was less pronounced after viral transduction, possibly somewhat overridden by the preference of γ-retroviral vectors for promoter-proximal integration  <cit>  and the resulting elevated background expression levels.
in any case, the improvements observed are solely due to the newly designed tet operator heptamer with  <dig> nt spacing between the centers, as the hcmv minimal promoter  remained unaltered.
so far, the relative contribution of spacer length variations versus alterations of nucleotide sequences separating them remains unclear.
changes in the phasing of bound transactivators cannot account for different background expression levels, only for differences in activated expression.
it is interesting to note, that for the available  <dig> nt spaced operator constructs  varying induction levels were obtained compared to a  <dig> nt operator spacing in the context of otherwise identical promoters.
in contrast to our own results  showing an increased inducible expression level with the  <dig> nt spaced operators, the study of agha-mohammadi showed a reduction in induced expression .
so far we do not have an explanation for these differences.
the first step towards improving the regulatory properties of the cmv minimal promoter was the introduction of a high affinity tfiib binding site  upstream of a consensus tata-box into ptet-t <dig>  this, other than the changes in the operator heptamer, led to a reduction of background activity most pronounced after stable integration of the ptet-t <dig> by a retroviral vector.
these results are in line with published data that implicate a negative effect on basal transcription levels for such promoter modifications without affecting the activated levels of transcription  <cit> .
in addition, the composition of minimal promoters with respect to cis elements or the interaction of different acidic activation domains with tfiib may play a significant role in the process of non-induced and induced transcription, and might be modulated depending on the transacting factors available in a given cell type  <cit> .
to further reduce the unregulated background expression we truncated the 5'-utr of the cmv-minimal promoter.
deletions of promoter elements within the leader, e.g.
the dpe, have been shown to improve the signal to noise ratio of the minimal promoter  <cit> .
indeed, a direct comparison of ptet- <dig>  ptet-t <dig> and ptet-t <dig> with ptet- <dig> by transfection, revealed a strongly reduced background expression in the off-state for these three promoters .
thus, in the transient state the expected reduction of background expression by the bre became functional only after removal of the dpe element  <cit> .
a pronounced reduction of baseline expression of ptet-t <dig> and ptet-t <dig> vs. ptet- <dig> suggests that the suppressing effect of tfiib on background transcription was partly masked by the dpe element.
however, the effect of deleting the dpe might not be directly related to the function of the bre.
in case of ptet-t <dig> we relied on an unusually short 5'-utr.
given the importance of untranslated leader regions for the functionality of mrnas and reconciling their average length of around  <dig> nt  <cit>  we tried to increase their length without losing the gains in dynamic range.
in order to increase the size of the untranslated leader, a 5'-utr fragment of the turnip yellow mosaic virus , was fused downstream to the residual cmv sequence of ptet-t <dig>  this leader was chosen because we assumed that cis-elements of the plant kingdom will not or only by chance be effective in the mammalian background.
indeed, the unregulated background expression was further reduced by ptet-t <dig>  the elimination of two hairpins within the tymv 5'-utr fragment  <cit>  resulted in further reduction of background expression in the off-state of the tet-system.
this holds true for both, the transient and the stable approach, since ptet-t <dig> displayed a 50000-fold up-regulation of gene expression in the plasmid based system  and about 10000-fold after its stable integration by a γ-retroviral vector .
among all the different promoter designs tested, ptet-t <dig> was consistently found to be the optimal ptet promoter.
we are currently using this promoter routinely in our laboratories when setting up tet-responsive stable cell lines.
ptet-t <dig> promoter has been successfully tested in stable cell lines as diverse as hek <dig>  primary human fibroblasts and an avian cell line .
here we also showed that the dox dose response of this promoter was unaltered when compared to the original ptet- <dig>  allowing an efficient, titratable expression control even in transfected cell populations.
in many applications these improvements will allow tight control over transgene expression while omitting the time consuming step of clonal isolation.
however, our results for the comparative analysis of pooled vs. clonal isolates of erbb <dig> expressing cells indicated that, obviously, for the precise adjustment of intermediate level expression the pool strategy is not optimal.
indeed, pools showed rather heterogeneous expression levels under these induction conditions, most likely due to integration site effects with individual loci following their inherent partial induction schemes.
this would be expected from differences in e.g.
chromatin packaging.
considering the different causes responsible for background expression in the off-state, such effects have to be addressed by means other than optimizing ptet promoters for their lowest level of intrinsic background expression.
CONCLUSIONS
this study demonstrated the feasibility to further improve tetracycline responsive promoters, especially for reduction of background expression of the tet regulatory system in the off-state.
to confirm that the newly created ptet design is of general use, the promoters were analyzed in two different assay systems, namely following transient transfection or otherwise chromosomal integration.
given our limited understanding of many aspects of mammalian promoter architecture, modifications of cis elements will always be partly empirical.
the results presented here nevertheless show, that some of the promoter elements tested, display similar behavior whether analyzed in the context of ptet promoters or in their original promoter environment.
from our previous experience we are confident that these findings from different cell culture systems will hold up in animal experiments, although this has yet to be proven.
thus, we anticipate that these novel ptet promoters will contribute to further refining and enhancing the tet technology for in vivo and in vitro use.

 the tet system is the most widely used inducible gene expression technology in eukaryotes, for both, in vivo and in vitro applications. based on its original design, now frequently referred to as „tet-off" system, it experienced its first major modification by the introduction of the „tet-on" system. both systems respond in opposite ways to the presence of tetracyclines, by either inactivating or activating gene expression. the difference is based on amino acid exchanges in the synthetic transcription factors employed, tta and rtta respectively, reversing the response of the dna binding domain to the presence of the allosteric effector dox. initially both systems relied on the same composite tet-responsive promoter, ptet-. subsequent attempts to broaden the utility and performance of the tet system focused on transactivator manipulation: nuclear localization sequences were introduced, codon usage was optimized, potential splice sites were removed and activation domains have been exchanged. the most significant advances, however, came from genetic approaches to identify improved versions of tet-on type transactivators. these experiments aimed to improve the dynamic range, by either a reduction of residual dna binding in the non-induced state or enhancement of activation in the induced state. in this context, the most notable transactivator alleles were identified in yeast and, through enforced retroviral evolution, in human cells. while these approaches focused on improving the dynamic range of the tet system via modified transactivators, it appeared interesting to explore, whether the tet system could also be improved by manipulation of the cis-acting tet-responsive promoters. the initially introduced ptet- is based on a minimal promoter fragment derived from the cmv immediate early promoter. various alternatives were introduced over the years, like those further truncating the cmv minimal promoter of ptet-, constructs based on the hsv tk promoter, the mmtv long-terminal repeat promoter and the hiv- long-terminal repeat promoter. several of these ptet variants had their advantages under the specific conditions tested, with e.g. a "second-generation tetracycline regulatable promoter" showing up to orders of magnitude in inducible gene regulation experiments using the tet-off regulation principle. however, with the exception of ptet-, which was derived from ptet- by empirical modifications and displayed a substantially reduced background expression, none of these promoter constructs is widely used. here we present our results with a series of novel ptet variants. systematically introduced alterations were generated to minimize background expression while maintaining high levels of induced expression. to place these experiments in a proper context, it is worthwhile to initially distinguish between the different sources of such background expression. especially in a comparative analysis of transient transfection and stable transduction experiments as presented here, their individual contribution can be readily addressed. considering the various sources of background expression is an essential requirement when expression systems based on the transactivation paradigm are engineered and is not restricted to the tet system. in principle, background expression can be triggered by residual binding of transactivators to their cognate binding sites. this would be the case when expression, driven by a ptet promoter, is higher in a tet transactivator-positive cell line in the off-state as compared to an otherwise isogenic tet transactivator-negative cell line. secondly, background expression due to e.g. enhancers or other sequence elements able to act at long-range on ptet is to be expected and has indeed been frequently observed. such effects can be avoided by targeted insertion of the ptet driven transcription unit into an appropriate genomic locus or suppressed by the use of insulator elements. thirdly, background expression is also influenced by the accessibility of the ptet, which is determined by epigenetic parameters such as dna methylation and integration into chromatin structures. lastly, background expression might be intrinsic for a given ptet due to hidden binding sites for transcription factor. reduction of this intrinsic background was the goal of the current study. for this purpose, we designed novel ptet promoter variants with low background activities and highly induced expression levels by stepwise modification of ptet- already known functional cis elements of the promoter where exchanged, others expected to optimize its performance were introduced and those suspected to impede performance where deleted. this strategy proved successful when the new ptet promoter variants where quantitatively characterized in a plasmid vs. retrovirus backbone and examined in different cell lines. our results revealed that several novel ptet promoters, and in particular, ptet-t are superior to ptet- and ptet- with regard to low background levels in the off-state, while retaining a high activation potential.  experimental strategy the tet system with its key elements ptet and the tetracycline controlled transcriptional activators is outlined in figure 1a. ptet- is composed of a cmv derived minimal promoter linked downstream to an array of seven tet operators. the focus of this work was the further optimization of ptet- via reduction of background expression while maintaining or even improving the levels of induced expression. such combined effects would result in an improved range of transcription control. for comparison ptet- and ptet- were included in the study. as outlined in figure 1b, these two promoters differ in the spacing between tet operators. the center-to-center distance of neighboring operators is nt for ptet- versus nt for ptet- moreover, their respective cmv minimal promoter moieties differ in length. the t-series of ptet promoters introduced here all contain a tet operator heptamer with nt spacing. in contrast to ptet- where the spacer sequences between the operators are identical we used randomized spacer sequences, thus minimizing the symmetry within the operator region. this accounts for the sensitivity of viral vectors to palindromic sequences due to the strand displacement mechanism, which may lead to gradual loss of operators during reverse transcription. replacing the operator heptamer of ptet- with this newly designed operator array resulted in ptet-t subsequent modifications of the minimal promoter moiety of the ptet promoters followed a rational design strategy, stepwise altering known functional sequence elements within the cmv minimal promoter as used in ptet-t introducing point mutations to ptet-t led to consensus sequences of the tata-box and the tfiib binding site in ptet-t the bre upstream the tata-box could possibly enhance transcriptional initiation upon induction with acidic domain-type transactivator vp, while reducing background transcription of promoters in the non-induced state. deletion of the cmv 5'-utr led initially to ptet-t with truncated downstream promoter element. ptet-t exhibiting a rudimentary 5'-utr, contained only the cmv-initiator element directly followed by the start codon. in order to restore an average length 5'-utr, we decided to introduce an unrelated eukaryotic sequence from a plant rna virus fused 3' to the inr sequence of ptet-t this sequence contains only few cis elements known to be important for promoter regulation in the animal kingdom. the transcription factor binding sites found, for rfx- and ap- were eliminated by point mutations, resulting in ptet-t further modifications of the tymv 5'-utr led to ptet-t and ptet-t as discussed in more detail below, these constructs are devoid of potential splice signals and hairpin structures that could confer cap-independent translation initiation. we were primarily interested in functional properties of the newly designed ptet promoters in heterogeneous, non-clonal cell populations. we therefore evaluated these promoters by transient transfection of plasmid dna, as well as γ-retroviral transduction resulting in stable cell pools. initial characterization of the new promoters in a plasmid backbone the new ptet promoters as well as the control constructs, ptet- and ptet-14) were all integrated in an identical manner into the plasmid puhc-131- containing firefly luciferase as the reporter gene. transfection into tet transactivator-positive hela-em cells was performed in the presence and absence of dox. the results as shown in figure demonstrate that all the new ptet promoters were highly inducible. ptet-t displayed a 5-fold higher induced expression level than ptet- although both contain the identical cmv minimal promoter. ptet-t also displayed a 6-fold reduction of non-induced background expression resulting in a regulation factor of at least 13000-fold vs. 400-fold for the original ptet- this improvement is solely based on the new operator heptamer introduced into otherwise identical constructs. the expression characteristics of ptet-t were similar to that of ptet-t differing only by the introduction of a consensus bre in the t construct. modifications of the 5'-utr led to slightly reduced expression in the induced state when compared to ptet-t still, even the weakest of these ptet promoters, ptet-t displayed a slightly higher induced expression level than the original ptet- in line with our experimental objective, the more interesting outcome of the stepwise alteration of the minimal promoter when modifying the 5'-utr was the further reduction of background expression in the off-state, already obvious from shortening the cmv leader sequence in ptet-t and ptet-t. introduction of the tymv 5'-utr and its variants reduced background expression even further. especially ptet-t displayed significantly lower background activity, more than 100-fold reduced compared to ptet- and about 10-fold compared to ptet- this resulted in regulation factors of about 50000-fold in these transient transfection assays. it should be noted here that for several of the ptet promoters the luciferase values measured by us were very close to instrument background. compared to our established protocols we increased the amount of transfected plasmid dna in order to obtain readings that could be reliably quantified. performance of the new regulatory units after retroviral transduction the newly designed ptet promoters were integrated into the es. γ-retroviral vector containing lmg* as the reporter gene. lmg* is a dual reporter gene providing gfp as well as luciferase activity. this allowed us to determine differences in unregulated background expression with exceptional sensitivity in luciferase assays without sacrificing the option of single cell resolution via gfp measurements. infections of htta- cells were performed at low moi under induced conditions. this ensured that the majority of the transduced cells contained a single integrated copy of the response unit. the results shown in figure 3c were similar but not identical to those of the transfection experiments. all new ptet promoters displayed high expression levels in the induced state, slightly higher than ptet- a direct comparison of reporter activities in absolute terms is not possible between the different experiments, as we had to switch from luciferase activity normalized to a second co-transfected reporter to luciferase activity normalized to the protein content of cellular extracts. however, the pattern of background expression levels in the off-state of the system were similar between the different ptet promoters when comparing the results shown in figure with those of figure as in case of the plasmid transfections the ptet-t transduced cell populations displayed the lowest background expression after chromosomal integration. the only notable difference resulted from the introduction of the bre, which had a much more pronounced effect on background expression upon chromosomal integration. comparison of the doxycycline dose response of ptet- and ptet-t6 next, we asked whether the altered ptet promoter architecture would result in dose response changes of the regulatory system towards dox. these parameters were previously established for ptet- in hela cells. hela-em cells were transduced by es.1-ptet-1p or es.1-ptet-t6p retroviral sin-vectors. purified populations were incubated in the presence of the indicated dox concentration for hours, harvested and analyzed for egfp fluorescence and luciferase actvity. the gfp data confirm that ptet-t can reach a slightly improved expression level when compared to ptet- since in the non-induced state, the mean fluorescence of both cell populations is too close to the background level of hela-em cells no meaningful conclusion can be drawn. taking advantage of the luciferase activity of the lmg* reporter, simultaneous luciferase measurements revealed that background expression in the off-state of ptet-t is 15-fold below that of ptet- in line with the previous analysis. the combination of reduced background and slightly enhanced induced expression level resulted in an about 30-fold increased regulation range. the maximum induction for both promoters had been reached at ng dox/ml; further increase of the effector concentration was without effect. note that the steep increase of gene expression between and ng dox/ml is similar for both ptet promoters, indicating an identical synergistic response of both promoters to increased occupancy of the operators by tet-transactivators. the facs analysis at single cell resolution indicates that all cells responded to induction, but not homogenously. upon partial induction the expression level of the individual cells differed widely. it has to be emphasized that the cell population analysis was genetically heterogeneous with respect to insertion of the reporter unit. again, the overall characterization of the two ptet promoters analyzed revealed no major differences in the dose response parameters. fine-tuning of transgene expression the results obtained with the new ptet promoters, especially ptet-t led us to routinely employ these promoters in ongoing research projects in our laboratories. the following results were derived from a project aiming at the conditional expression of erbb in breast cancer cell lines. a tet transactivator-positive mda-mb ro cell line expressing rtta-s has been previously established. transduction of this clone was performed with a retroviral sin-vector, es.1-t6p, in which the erbb open reading frame was under control of ptet-t as the erbb protein is located on the cell surface, it is well suited for detection and selection of the transduced population by facs. the results of the cell-based analysis of the purified population showed, that erbb expression was tightly regulated in these experiments with no recognizable background expression in the off-state, despite looking at non-clonal pools expression could be adjusted to intermediate levels by titrating dox, and cells could be maintained at a chosen expression level over a prolonged period of time. this time course analysis was terminated on day interestingly, prolonged full induction led to the disappearance of erbb cells, showing highest expression levels, as can be seen from the asymmetric steep drop of the right shoulder of the expression profile peak. thus, the expression level appears to reflect the maximum long-term expression level of erbb tolerated by those cells. the only other recognizable difference in the expression profiles was a slight „compression" of intermediate expression at ng/ml dox, as the cell-to-cell variability in transgene expression was apparently reduced after prolonged growth under these conditions. given the non-clonal origin of this cell population, individual cells behaved remarkably similar. still, for certain applications, like analyzing pharmacological responses to varying densities of cell surface receptors, the homogeneity under intermediate expression conditions may not be sufficient for reliable results. thus, we examined whether respective homogeneous cell populations, derived from cell clones, would improve the situation. the example shown in figure 5b, lower panel, demonstrates that this is indeed the case. further suppression of background expression could already be observed in the vast majority of cells, but the precision of partial induction was markedly improved by clonal isolation of cells harboring ptet-t discussion our goal was to improve the dynamic range of tetracycline regulated transcription in such a way that tight, virtually background-free non-induced expression can routinely be obtained when working with non-clonal cell populations. the tet system in its current configuration has proven its capacity to achieve such background free expression, as widely documented in the literature. however, most of these studies relied on clonal analysis of transgenic cells, thereby eliminating cell clones with less favorable properties, an often laborious process which at times might even be impractical, for example in the genetic engineering of primary cells. by using previously established and well-characterized transactivator-positive cell lines we restricted our efforts solely to cis elements of tet-controlled transcription units, the tet-responsive promoters. we investigated if gene expression regulated by a rationally designed series of new ptet promoters would be superior over the widely used original ptet- promoter and its derivative ptet-, particular by further reducing background expression of tet-regulated transcription units while maintaining fully-induced expression levels. we conclude that the reduction of the ptet background expression reported here is exclusively due to the low intrinsic background of these new tet-responsive promoters. their side-by-side analysis in different established tet transactivator-positive cell lines eliminated the possibility that effects like transactivator abundance or differences in tta-vs. rtta-mediated transcription control would influence our conclusions. moreover, the tet operator sequences themselves are identical in all new ptet promoters used and any potential residual transactivator affinity to the operators would not have any influence on the outcome of our study. lastly, we excluded a systematic distortion of our results by the possible occurence of integration site-specific or epigenetic effects through parallel analysis of all ptet promoters in transient transfection assays. the initial transition from ptet- to ptet-t resulted in a more than 5-fold lower background expression in transient transfection assays. this effect was less pronounced after viral transduction, possibly somewhat overridden by the preference of γ-retroviral vectors for promoter-proximal integration and the resulting elevated background expression levels. in any case, the improvements observed are solely due to the newly designed tet operator heptamer with nt spacing between the centers, as the hcmv minimal promoter remained unaltered. so far, the relative contribution of spacer length variations versus alterations of nucleotide sequences separating them remains unclear. changes in the phasing of bound transactivators cannot account for different background expression levels, only for differences in activated expression. it is interesting to note, that for the available nt spaced operator constructs varying induction levels were obtained compared to a nt operator spacing in the context of otherwise identical promoters. in contrast to our own results showing an increased inducible expression level with the nt spaced operators, the study of agha-mohammadi showed a reduction in induced expression. so far we do not have an explanation for these differences. the first step towards improving the regulatory properties of the cmv minimal promoter was the introduction of a high affinity tfiib binding site upstream of a consensus tata-box into ptet-t this, other than the changes in the operator heptamer, led to a reduction of background activity most pronounced after stable integration of the ptet-t by a retroviral vector. these results are in line with published data that implicate a negative effect on basal transcription levels for such promoter modifications without affecting the activated levels of transcription. in addition, the composition of minimal promoters with respect to cis elements or the interaction of different acidic activation domains with tfiib may play a significant role in the process of non-induced and induced transcription, and might be modulated depending on the transacting factors available in a given cell type. to further reduce the unregulated background expression we truncated the 5'-utr of the cmv-minimal promoter. deletions of promoter elements within the leader, e.g. the dpe, have been shown to improve the signal to noise ratio of the minimal promoter. indeed, a direct comparison of ptet- ptet-t and ptet-t with ptet- by transfection, revealed a strongly reduced background expression in the off-state for these three promoters. thus, in the transient state the expected reduction of background expression by the bre became functional only after removal of the dpe element. a pronounced reduction of baseline expression of ptet-t and ptet-t vs. ptet- suggests that the suppressing effect of tfiib on background transcription was partly masked by the dpe element. however, the effect of deleting the dpe might not be directly related to the function of the bre. in case of ptet-t we relied on an unusually short 5'-utr. given the importance of untranslated leader regions for the functionality of mrnas and reconciling their average length of around nt we tried to increase their length without losing the gains in dynamic range. in order to increase the size of the untranslated leader, a 5'-utr fragment of the turnip yellow mosaic virus, was fused downstream to the residual cmv sequence of ptet-t this leader was chosen because we assumed that cis-elements of the plant kingdom will not or only by chance be effective in the mammalian background. indeed, the unregulated background expression was further reduced by ptet-t the elimination of two hairpins within the tymv 5'-utr fragment resulted in further reduction of background expression in the off-state of the tet-system. this holds true for both, the transient and the stable approach, since ptet-t displayed a 50000-fold up-regulation of gene expression in the plasmid based system and about 10000-fold after its stable integration by a γ-retroviral vector. among all the different promoter designs tested, ptet-t was consistently found to be the optimal ptet promoter. we are currently using this promoter routinely in our laboratories when setting up tet-responsive stable cell lines. ptet-t promoter has been successfully tested in stable cell lines as diverse as hek primary human fibroblasts and an avian cell line. here we also showed that the dox dose response of this promoter was unaltered when compared to the original ptet- allowing an efficient, titratable expression control even in transfected cell populations. in many applications these improvements will allow tight control over transgene expression while omitting the time consuming step of clonal isolation. however, our results for the comparative analysis of pooled vs. clonal isolates of erbb expressing cells indicated that, obviously, for the precise adjustment of intermediate level expression the pool strategy is not optimal. indeed, pools showed rather heterogeneous expression levels under these induction conditions, most likely due to integration site effects with individual loci following their inherent partial induction schemes. this would be expected from differences in e.g. chromatin packaging. considering the different causes responsible for background expression in the off-state, such effects have to be addressed by means other than optimizing ptet promoters for their lowest level of intrinsic background expression.  this study demonstrated the feasibility to further improve tetracycline responsive promoters, especially for reduction of background expression of the tet regulatory system in the off-state. to confirm that the newly created ptet design is of general use, the promoters were analyzed in two different assay systems, namely following transient transfection or otherwise chromosomal integration. given our limited understanding of many aspects of mammalian promoter architecture, modifications of cis elements will always be partly empirical. the results presented here nevertheless show, that some of the promoter elements tested, display similar behavior whether analyzed in the context of ptet promoters or in their original promoter environment. from our previous experience we are confident that these findings from different cell culture systems will hold up in animal experiments, although this has yet to be proven. thus, we anticipate that these novel ptet promoters will contribute to further refining and enhancing the tet technology for in vivo and in vitro use. 
in this study, we systematically modified the widely used cytomegalovirus minimal promoter to further minimize background expression, resulting in an improved dynamic expression range. they are indispensable to warrant low expression levels with the system turned "off". using both plasmid-based and retroviral gene delivery, our analysis revealed that especially background expression levels could be significantly reduced when compared to previously established "standard" promoter designs. our results also demonstrate the possibility to fine-tune expression levels in non-clonal cell populations. the performance of the tetracycline controlled transcriptional activation system depends critically on the choice of minimal promoters. nevertheless, the partly empirical modification of cis-elements as shown in this study can lead to the specific improvement of the performance of minimal promoters. the novel composite ptet promoters introduced here will further expand the utility of the tet system. on the other hand, they must support high level of gene expression in the "on"-state.
 the performance of the tetracycline controlled transcriptional activation system depends critically on the choice of minimal promoters. they are indispensable to warrant low expression levels with the system turned "off". on the other hand, they must support high level of gene expression in the "on"-state.  in this study, we systematically modified the widely used cytomegalovirus minimal promoter to further minimize background expression, resulting in an improved dynamic expression range. using both plasmid-based and retroviral gene delivery, our analysis revealed that especially background expression levels could be significantly reduced when compared to previously established "standard" promoter designs. our results also demonstrate the possibility to fine-tune expression levels in non-clonal cell populations. they also imply differences regarding the requirements for tight regulation and high level induction between transient and stable gene transfer systems.  until now, our understanding of mammalian transcriptional regulation including promoter architecture is limited. nevertheless, the partly empirical modification of cis-elements as shown in this study can lead to the specific improvement of the performance of minimal promoters. the novel composite ptet promoters introduced here will further expand the utility of the tet system. 