Graded or threshold response of the tet-controlled gene expression: all depends on the concentration of the transactivator
© Niels et al.; licensee BioMed Central Ltd. 2013
Received: 17 August 2012
Accepted: 14 January 2013
Published: 22 January 2013
Currently, the step-wise integration of tet-dependent transactivator and tet-responsive expression unit is considered to be the most promising tool to achieve stable tet-controlled gene expression in cell populations. However, disadvantages of this strategy for integration into primary cells led us to develop an “All-In-One” vector system, enabling simultaneous integration of both components. The effect on tet-controlled gene expression was analyzed for retroviral “All-In-One” vectors expressing the M2-transactivator either under control of a constitutive or a new type of autoregulated promoter.
Determination of luciferase activity in transduced cell populations indicated improvement of the dynamic range of gene expression for the autoregulated system. Further differences were observed regarding induction kinetics and dose–response. Most notably, introduction of the autoregulated system resulted in a threshold mode of induction, whereas the constitutive system exhibited pronounced effector-dose dependence.
Tet-regulated gene expression in the applied autoregulated system resembles a threshold mode, whereby full induction of the tet-unit can be achieved at otherwise limiting doxycycline concentrations.
In this study, we combined the key benefits of the self-contained and the autoregulated system with a bidirectional vector design. The two vectors explored in this study differed regarding their mode of transactivator expression. In the “self-contained” MOV-scT6 vector, M2 transactivator expression is under control of the human PGK promoter , while in the “autoregulated” MOV-scT6cA vector M2 transactivator expression is driven by the newly developed synthetic "cA" promoter, a weak constitutive but inducible minimal promoter. Selected cell populations were used to compare the regulatory properties of both vectors with respect to their effector dose–response and kinetics of activation.
Design of the bidirectional vectors
Two MOV-vectors were constructed, “self-contained” (MOV-scT6) and “autoregulated” (MOV-scT6cA), where M2-transactivator expression was either placed under control of the constitutive human phosphoglycerate kinase promoter (hPGK), or a newly designed tet-responsive “cA”-promoter (Figure 2B, see below). MOV vectors also contained a shortened version of the woodchuck hepatitis virus posttranscriptional regulatory element (pre*s; Additional file 1: Figure S2). M2 transcripts were terminated at the pA signal of the viral 3´-LTR.
Properties of the regulatory unit within monocistronic vectors and the self-contained bidirectional vector
Expression level and regulatory potential of unidirectional and bidirectional vectors
4.1 ± 0.6
4.1 ± 0.1
9.8 ± 1.5
1.9 ± 0.2
4.7 ± 0.6
4.0 ± 0.8
1.2 ± 0.01
9.2 ± 2.0
1.3 ± 0,3
Although the dynamic range of gene regulation was shown to exceed previously published One-vector systems, further improvement was necessary to obtain full induction.
Replacement of the constitutive PGK-promoter by an artificial inducible-promoter
To improve vector performance, we developed a minimal promoter designed to inhibit weak constitutive as well as inducible activity, thus introducing the autoregulated principle into bidirectional vectors. The newly designed cA promoter (Figure 2A, Additional file 2: Figure S1) consists of an HIV-1 minimal promoter, with low background activity in the context of a TRP  fused to the CAAT-box of the MoMuLV-LTR promoter. The latter was shown to be sufficient to provide residual activity of a minimal LTR . This promoter was designed (i) to minimize crosstalk with the TRP, and (ii) to guarantee low basal levels of M2 transactivator during the “off-state”, while being sufficiently active to initiate the positive feedback loop. Replacing the PGK by the cA-promoter resulted in the generation of MOV-scT6cA vector (Figure 2A), which was considered to be autoregulated, providing a low constitutive activity for M2 transactivator expression.
Increasing gene dosage (Figure 3A, right) strongly enhanced gene expression upon induction (up to 108 rlu/μg), while the dynamic range of gene regulation was reduced. This phenomenon was observed in both vector systems. Based on luciferase data, a reduction in background activity could only be demonstrated for cell populations of the autoregulated vector, transduced at low MOI. This observation reveals the impact of the integration site, since under this condition variation due to position effect is pronounced.
Furthermore, severe effects on cell growth were observed for the autoregulated vector system, when cells were treated with high gene dosage, followed by induction (Additional file 3: Figure S3). This effect can most likely be attributed to high transactivator abundance and hence squelching.
Autoregulation altered the mode of induced gene expression
As generally accepted, tet-controlled gene expression enables effector-dose dependent adjustment of transgene steady state levels. Therefore, dose–response experiments were performed to further characterize the two construction principles.
Further differences between the two vectors were revealed by FACS-based analysis of enriched Ht1080 cell populations (Figure 4B). As expected, transgene expression was found to be effector dose-dependent in cells transduced by the self-contained MOV-scT6 vector, with considerable intermediate levels at 10–100 ng Dox/ml. In contrast, full induction rates were observed at already lower effector concentrations for the autoregulated MOV-scT6cA vector and further increase in effector (Dox) concentration resulted only in increased numbers of induced cells (Figure 4C). Therefore, kinetics of the autoregulated vector rather resembled a threshold mode.
Taken together, tet-regulated transgene expression was found to resemble a threshold mode in the autoregulated system. Following the law of mass action, full induction rates depended on the concentrations of M2-transactivator and its ligand (Dox), respectively. Variations observed at single cell level indicated insufficient M2-transactivator levels for a subset of the transduced cells. Since this could be overcome in systems were transactivator was provided from an independent locus, basal activity of the cA-promoter rather than of the TRP had been affected at the integration site.
Induction kinetics of self-contained and autoregulated vectors
This important difference is further illustrated in Figure 7C. While about 60% of the cells transduced by the self-contained MOV-scT6 vector showed clear induction after about 4 hours, only about 10% of the population transduced by the autoregulated MOV-scT6cA vector displayed a fast response. During further induction, the percentage of induced cells increased only slowly compared to the rapid activation of all cells transduced by the self-contained MOV-scT6 vector, suggesting involvement of particular cellular events, which influence the chromosomal environment and thereby the activity of the TRP/cA promoter.
Since the mid 90‘s, numerous studies have explored strategies for simultaneous (and reliable) transfer of both tet-system components into target cells. However, achieving tight control in “One-vector systems” has remained a challenge, as the dynamic range of gene expression was found to be hampered by high background and/or low transgene expression. In this study, we report on the design of a new MoMuLV-based One-vector system, with promising features. Firstly, open reading frames of the two components were expressed bidirectionally. Overlapping transcripts can thus be avoided, as these might reduce expression levels and negatively effect the dynamic range of tet-regulated gene expression [20, 27, 29]. Secondly, expression of the M2-transactivator was driven by the newly designed “cA” promoter, which exhibited weak basal as well as inducible activity. Results obtained from Ht1080 cell populations transduced with either the newly designed autoregulated vector, MOV-scT6cA, or the self-contained vector, MOV-scT6, demonstrate the superiority of the developed One-vector system (Figure 3). While both vectors showed high inducible expression, based on luciferase activity (bulk assay), the dynamic range of gene regulation in the autoregulated MOV-scT6cA vector was found to be increased by 3.7-fold as compared with the self-contained MOV-scT6 vector (4.8×103 vs. 1.3×103-fold). This improvement was largely due to the reduced background activity in the autoregulated MOV-scT6cA vector. Our results further suggest that promoter interference  between the tet-responsive Ptet-T6 and cA-promoter was reduced compared to the combination of Ptet-T6 and PGK-promoter and that a selection for integration sites promoting basal activity of the TRP/cA-promoter did not occur. These observations are in accordance with the findings of Lindemann and co-workers , who reported best results for an autoregulated MoMuLV-based system with respect to expression levels and regulatory properties in vitro and in vivo, when transactivator expression was driven by an enhancer-deleted LTR. Functionality of the cA-promoter design was further demonstrated by analysis of the M2-mRNA steady state level in the absence of Dox, revealing a 50% reduction compared to the PGK-promoter (Figure 5B). Infection of cell populations at increasing MOIs led to enhanced expression levels of the dual reporter gene lmg*, demonstrating an increase in gene dosage. However, at high MOI (≥1), cell populations transduced by MOV-scT6cA displayed strong growth retardation under inducing conditions (Additional file 3: Figure S3), suggesting massive accumulation of M2-transactivator to levels that caused squelching [23, 34, 35]. The moderate growth retardation observed in cells transduced by MOV-scT6 might be explained by exhaustion of other essential cell components, e.g. amino acids or nucleotides, since here expression levels of the dual reporter gene lmg* went into extremes (>4×107 rlu/μg protein).
As expected, the dose–response analysis of the two vector types, self-contained (MOV-scT6) and autoregulated (MOV-scT6cA), revealed a significant difference in their response mechanism (Figures 4 and 5). While the self-contained vector exhibited a more graded, Dox-dependent induction of gene expression [36, 37], a threshold mode was observed for the autoregulated vector. This important difference was only detected at the single cell level, as demonstrated in cell based analysis of eGFP fluorescence of the dual reporter gene lmg*, since it was masked in luciferase analysis of bulk cultures.
Markusic and co-workers obtained similar results  by direct comparison of a self-contained and an autoregulated unidirectional lentiviral vector. In their study, populations transduced by the autoregulated vector displayed a nearly full induction of gene expression at yet intermediate effector (Dox) concentrations and an increase in positive cells at higher Dox concentration (Figure 5 in their paper). From the combined results it may be concluded that the threshold response was due to the autoregulated mode for transactivator expression. Further observations support the hypothesis that basal transactivator abundance might be the limiting factor: i) a sub population, enriched for its ability to achieve full induction levels at 30 ng/ml Dox, displayed an increased steady state level of M2-mRNA already before induction (Figure 5B), and (ii) Hela-EM2 cells, which provide a basal abundance of M2 transactivator, showed a threshold response of the total cell population at 30 ng/ml Dox, when transduced by the autoregulated MOV-scT6cA vector.
From these observations, a model following the law of mass action can be derived, with activation of transgene expression being proportional to the product of the concentrations of M2-transactivator and its effector Dox. Thus, full activation of the TRP-driven transgene could be achieved at low M2-transactivator levels, given that effector concentration remained at optimum level (Figures 4, 5; 1000 ng/ml Dox), or, vice versa, at high levels of M2-transactivator at otherwise limiting Dox concentrations (Figures 5, 6; 30 ng/ml Dox).
Our data further suggest that the basal activity of the cA-promoter is dependent upon the integration sites. Only loci that favoured the start of the autoregulated circuit were able to induce the threshold response of the Tet-system at low Dox concentrations. The accessibility of the TRP at the chromosomal integration site seems to be of minor importance for the conversion of the graded to a threshold response.
In summary, our results demonstrated the advantageous properties of the autoregulatory compared to the self-contained principle for M2-transactivator expression, when using retroviral vectors with a bidirectional design, combined with the inducible cA-promoter. However, limitations occur when high vector dosages are applied. In particular, the observed on/off switch may have significant advantages, especially considering that full activation was achieved at suboptimal Dox concentrations and thus might help to overcome induction problems related to tissue-specific barriers for effector penetration. However, graded induction of gene expression is not possible with the autoregulated cA promoter and thus excludes this promoter design from experiments where an adjustable mode of transgene expression is mandatory. Moreover, the dependence of induced gene expression on the cellular abundance of the transactivator provides important evidence to help explain the large difference of effector concentrations reported to fully activate TRPs in various cell systems.
293T (ATCC # CRL-11268), Hela-EM2  and Ht1080 cells were cultured in Dulbecco´s modified Eagles medium (DMEM, Invitrogen) supplemented with 10%, heat inactivated fetal bovine serum (FBS, PAA) at 5% CO2 and 37°C. Cultures were split at 70-80% confluency. Following a washing step with PBS and incubation for 3–5 min in the presence of PBS/EDTA (0,8 mM), cells were harvested and either transferred into fresh medium or used in subsequent analysis.
Transient vector production and titration
Transient production of viral vectors was carried out by lipofection with the TransIt293 reagent (Mirus, CA) as recommended by the supplier. About 1.5×106 293T cells were transferred to 60 mm dishes the day before transfection. A total amount of 15 μg plasmid DNA was transfected containing 5 μg pHIT60 (gag/pol expression plasmid; ), 5 μg pczVSV-G (VSV-G envelope expression plasmid ) and 5 μg of the transfer vector. 16–18 hours after transfection the medium was replaced by 3 ml DMEM-medium, supplemented with 5 mM Na-butyrate, which was exchanged for DMEM-medium without Na-butyrate after additional 6–8 hours. 16–18 hours following medium exchange the supernatant was harvested, filtrated (0,45 μm, Nunc), supplemented with polybrene (5 μg/ml, SIGMA), aliquoted and stored at −80°C for later use.
All titrations were performed on Ht1080 cells using serial dilutions of the obtained supernatants (5-10-20-40-80-160-fold, respectively). Briefly, 2×105 cells were transferred to a 6well plate the day before infection. 24 hours later medium was replaced by 1 ml of fresh culture medium supplemented with polybrene (5 μg/ml) and premixed with supernatant. After about 18–20 hours medium was renewed and cells were cultivated under induced conditions (Dox 1000 ng/ml). Fluorescence activated cell sorting (FACS) or otherwise analysis of cell populations were performed on day 6 (about 96 hours post induction). For calculation of viral titers the number of GFP positive cells (about 4×105 cells × % GFPpos/100) was determined, a correction factor of 2 was applied to account for cell division during infection. In general, titers in the range of 1-3×106 IP/ml could be obtained.
Establishing transduced cell populations
About 4×105 cells (Hela-M2) were infected (always in the absence of Dox) on 6well plates with serial dilutions of the transiently produced vectors and induced after the first split for four to five days at 1000 ng/ml Dox. Appropriate infected populations (1-3% positive cells) were used for the enrichment by one round of FACS. These conditions ensured, that mostly single copy integrates of the vectors were generated. In general, the established individual populations were adjusted to present >15.000 independent clones.
Determination of luciferase activity
Purified transduced cell populations had to be cultivated in the “off-state” for a period of least 10 days, due to the prolonged half life of luciferase in the fusion protein lmg* and the high expression level of the tet-units. Induction experiments were started by splitting 0.5-1×105 cells into cell culture medium with or without Dox (500 ng/ml). After 96 (72) hours incubation cells were harvested with PBS/EDTA and GFP fluorescence and luciferase activity were analyzed simultaneously. 0.5-2 μl of bulk cell lysate were used for analysis of luciferase activity by luminescence detection (Lumat, Berthold, Germany), essentially as described earlier . Protein concentration was determined according to the method of Bradford  and specific luciferase activity was calculated.
In general, treatment of cells was similar in dose response experiments, except for a daily medium exchange. This was applied in order to counteract the potential degradation of Dox, which may affect the level of induction especially at low concentrations. Medium was supplemented with the indicated Dox-concentrations.
Experiments on induction kinetics required transfer of individual cell numbers, thus, allowing the harvest of a sufficient amount of cells for short term cultures, and avoiding overgrowth of the cells used for prolonged cultivation. In general, cells for short term analysis (e.g. 0.5 hours of induction) were splitted to high density (5×105 cells/6well), while cells for the 24/48/72/96 hours induction were transferred at about 4-2-1 or 0.5 × 105 cells/6well.
Northern analysis of total RNA
For RNA analysis the enriched populations were grown on 9cm dishes either in the absence or presence of Dox. After 96 hours the cells were harvested and total RNA was extracted by the acidic phenol method . Northern analysis was performed as described earlier . Detection was carried out with avidin conjugated alkaline phosphatase (Molecular Probes) and CDP-Star (Tropix) as substrate for chemiluminescent detection. Rat GAPDH cDNA served as an internal mRNA standard. All probes used were biotin-labeled during PCR-synthesis. Detection of the mRNA steady states was achieved by exposure to X-Ray film (Kodak Bio-Max light, Sigma). Sizes of the RNA marker (Promega) are indicated in the figures. The following oligonucleotides were used for probe synthesis: sense 5´- TTACAGATGCACATATCGAGG, antisense: 5´-CCTCTGGATCTACTGGGTTA (rat GAPDH) and sense 5´- tctagactggacaagagc, antisense: 5’- ccgccgctttcgcactt (rtTA2s-M2). Densitometric analysis of appropriately exposed films was performed by use of NIH 1.57 software.
The retroviral SIN-vector “pES.1” used for the transfer of the tet-response units had been described earlier .
The inducible expression cassette consisted of a tet-operator heptamer, the Ptet-T6 TRP, the dual reporter gene lmg* and a modified (see below) posttranscriptional regulatory element of the woodchuck hepatitis virus (WPRE, ). While the transcription of the ES.1-T6 vector was terminated at the pA-signal of the viral 3-LTR, the ES.1-T6sc transcripts were terminated at the antisense orientated SV40(late) polyadenylation signal fused to the constitutive transport element (cte) of SRV-1 [32, 46]. The tet-responsive promoter as all other components was subcloned into pBluescript SKII+ plasmid backbone (Stratagene, CA) by standard techniques  and sequenced (Eurofins, Germany).
The WPRE element, which already contained mutations of “atg´s” of the original element, was newly synthesized by PCR, using the SIN11 vector  as template. Sequence alignment to the WPRE used in the lentiviral vectors of the Naldini Lab  showed a 400 bp homologous stretch. This sequence, common to both WPRE elements, was PCR amplified and used for generation of the constructs (Additional file 1: Figure S2).
The cA-promoter was PCR amplified using the S2f-clHCg  as template. The CAAT-box of MoMuLV was introduced upstream of the SP-1 sites by amplification with the particular sense oligo. The full sequence is given in Additional file 2: Figure S1.
We wish to gratefully acknowledge the help of Angelika Lehr and Michael Morgan for editing the manuscript and Thomas Rausch for inspiring discussion. We also thank Prof. Christopher Baum (Experimental Haematology, Hannover Medical School, Germany) for his support. N.H. is working in the Department of Experimental Haematology (Hannover Medical School). This work was supported by grants of the German ministry for Research and Education (CB-Hermes, 01GN0930) and the Deutsche Forschungsgemeinschaft (KL 1311/4-1 and Cluster of Excellence REBIRTH Exc 62/1).
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