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. 2001 Aug 15;29(16):e76. doi: 10.1093/nar/29.16.e76

Conditional cell ablation by stringent tetracycline-dependent regulation of barnase in mammalian cells

Stefanie Leuchtenberger, Angela Perz, Christiane Gatz 1, Jörg W Bartsch a
PMCID: PMC55860  PMID: 11504884

Abstract

Conditional expression of suicide genes in vivo has a wide range of applications in biological research and requires a minimal basal promoter activity in the uninduced state. To reduce basal activity of tetracycline (tc)-inducible target promoters we combined synthetic tet operators in varying numbers with a core promoter derived from the plant viral 35S promoter. An optimized promoter, PTF, was found to exert a stringent regulation of luciferase in combination with tTA and rtTA in different mammalian cell lines. We linked PTF to the barnase gene, coding for a highly active RNase from Bacillus amyloliquefaciens. Stable cell clones expressing barnase under control of tTA exerted cell death only after tc withdrawal, correlating with a 10-fold induction of barnase mRNA expression. Directing tTA expression through a neuron-specific enolase promoter (PNSE) leads to barnase expression and cell death in neuronal cells after tc withdrawal. Taken together, our data demonstrate that a stringent control of barnase expression in the uninduced state improves cell ablation studies, as high frequencies of transgene propagation in both cell lines and in transgenic mice are observed.

INTRODUCTION

For many applications in gene therapy and medical research, the expression of a suicide gene at defined time points is of great advantage. In particular, the cell-specific induction of toxin genes represents a promising approach for the functional analysis of distinct cell types in situ. In many human diseases, loss of distinct cell types leads to detrimental diseases, for instance in the central nervous system (CNS). Until now, conditonal cell ablation has been achieved either by introduction of the herpes simplex thymidine kinase (HSV-tk) cassette for functional analysis of astrocytes in transgenic mice (1,2) or by tetracycline (tc)-dependent expression of diphteria toxin A chain (DTA) in cell lines (3) and transgenic mice (4). These approaches faced several limitations: the HSV-tk gene is only applicable to ablation of proliferating cells within the CNS. Tetracycline controlled expression of DTA led to very low yields of recombinant cell clones of transgenic mice, as the original target promotor (5) revealed leakiness in the uninduced state.

The tc-dependent expression system is based on a chimeric transcriptional transactivator tTA, which is a fusion between the bacterial Tet repressor (TetR) and the constitutive activation domain of herpes simplex protein 16 (VP16). Binding of tTA to a target promoter consisting of multiple operators upstream of a core promoter leads to activation of transcription, which can be abrogated by addition of tc or the commonly used analog doxycycline (dox), which interferes with DNA binding of tTA. In addition, a TetR mutant that binds DNA only in the presence of tc has been exploited for the construction of rtTA, yielding a tc-inducible promoter (6).

In previous reports it has been demonstrated that the efficiency of the tc system is dependent on the cell line tested and is even more complex in transgenic mice where residual activity, even in the absence of the transactivator, depends on the tissue (7,8). The difference in expression levels between induced and uninduced transgenic tissue is 100 in optimal cases and varies in different tissues. Residual activity might be due to transcription factor binding to the CMV core promoter (7). The problem of leakiness is even more pronounced when rtTA is used as a transcriptional activator.

Three different approaches have been undertaken to reduce leakiness of the tc-dependent expression systems. Most recently, Urlinger et al. (9) isolated new rtTA alleles yielding lower activation in the uninduced state. Forster et al. (10) and Imhof et al. (11) used rtTA to drive tc-inducible expression of a suitable target promoter which was at the same time repressed by tTA in the absence of tc. This sophisticated arrangement reduced background activity by a factor of six, but has the disadvantage of requiring the simultaneous expression of two trans factors. Hoffmann et al. (12) used a modified mouse mammary tumor virus promoter (ΔMTV) in which the steroid-responsive elements were replaced by tetO sequences. However, as access of tTA depends critically on chromatin (13), induction levels are on average 10-fold lower than in the CMV based target promoter.

In this work we constructed a new target promoter with reduced leakiness, by exchanging the viral cytomegalovirus core promoter by a plant viral promoter and by avoiding identical spacer sequences between the multimerized operators. Promoter constructs were tested for basal activity either with tTA or with rtTA in different mammalian cell lines. As our major goal is to ablate neuronal cell types within the mouse CNS, we included in our analyses cell lines representing the rodent nervous system. To assess the usefulness of the optimised promoter in cell ablation experiments, we combined it with the lethal RNase gene from Bacillus amyloliquefaciens (barnase; 14), thus demonstrating a stringent cell ablation strategy for mammalian cells and tissues.

MATERIALS AND METHODS

Plasmids and cloning

The promoters PTAX and PTF were from plasmids pBHTAX and pBHTF (15). For cloning, the promoter sequences were amplified by PCR using primers with HindIII linkers. As control plasmids for tc-dependent luciferase expression, plasmids pXTC7C53Luc (PXTC-Luc; 5) and pMtetO5Luc (PMtetO5-Luc; 12) were used. The plasmids pTAX-Luc and pTF-Luc were obtained by digesting pMtetO5Luc with HindIII, and ligation of the HindIII promoter fragments into the respective vector sites. For construction of an expression vector containing the tc-inducible cDNA for Barnase, PTF was fused to the Barnase coding region (16). Addition of a mammalian polyadenylation signal was achieved by cloning it into the expression vector SBC-1 (17) using ClaI and HindIII. As a regulator plasmid, we used pUHD15-1 (5) or pUHD17-1 (6) encoding the tc-dependent transactivator (tTA or rtTA, respectively) under control of a human cytomegalovirus (hCMV) promotor (PCMV–tTA or PCMV–rtTA). For cell-specific expression of tTA, the hCMV promotor was removed by cleaving with XhoI, filled-in with Klenow DNA Polymerase and digested with EcoRI. A 1.8 kb neuronal-specific enolase (NSE) promotor-fragment was excised from pCRToPo-cloning vector (Invitrogen, Heidelberg, Germany) with EcoRI and PvuII and ligated into unique restriction sites of pUHD15 to construct the neuronal regulator plasmid PNSE–tTA.

The plasmids pRSVLacZII and pEGFP-N3 (Clontech, Heidelberg, Germany), containing β-galactosidase cDNA (lacZ) or GFP under control of constitutive promotors, respectively, were used as reporter plasmids.

Cell culture and transfections

HeLa (ATCC CCL 2), CHOdhfr– (ATCC CRL 9096) hamster ovary fibroblasts, C6 rat glioma cells (ATCC CCL 107), 293T human embryonal kidney cells (provided by Dr Jan Cornelis, DKFZ, Heidelberg, Germany), PC12 rat adrenal pheochromocytoma (ATTC CRL 1721), mouse motoneuron-like cell line NSC-19 (18), and Neuro-2A neuroblastoma cells (ATCC CCL 153) were maintained in Dulbecco’s modified Eagle medium (DMEM) containing 10% (v/v) foetal calf serum (FCS; Biochrom KG, Berlin, Germany); CHOdhfr– cells were cultured in the presence of Prolin (5 mM) and HT supplement (0.1 mM hypoxanthin, 0.01 mM thymidine). The cell line CHOAA8 (Clontech, Heidelberg, Germany), stably expressing the tc-dependent regulator tTA (plasmid pUHD15-1neo), was cultured in α-MEM supplemented with 10% FCS and 100 µg/ml G418 (Gibco BRL). Neuronal cell lines and C6 cells were transfected with Lipofectamine (Promega, Madison, WI), CHOdhfr– and 293T cells by using the calcium phosphate method. The transfection efficiencies were usually >20%, as determined by control transfections with pEGFP. Depending on the cell type, 1–3.5 × 105 cells were seeded into 30 mm plates and constructs were added 24 h later in the absence or presence of dox (Sigma, Steinheim, Germany). The dishes with dox were preincubated with 1.0 µg/ml 24 h before transfection. Transient cotransfections were performed with ratios of 5:1 regulator plasmid to target plasmid (luciferase or barnase) and cells were analysed 48–72 h after transfection. Luciferase constructs were stably integrated by electroporating 10 µg PvuI linearised plasmid in a Bio-Rad electroporator with 200 V and 480 µF. For stable transfection of barnase constructs, cotransfections were done in 1:10 and 1:20 ratios of pBSΔpac (CHOAA8) or pEGFP-neo (Neuro-2A) to PTF–barnase. Three days after transfection, transfectants were selected either with 3.5 µg/ml puromycin or 400 µg/ml G418 (Luciferase plasmids and pEGFP-neo).

Luciferase and β-galactosidase assays

Forty-eight hours after transfection, cells were lysed by the addition of 150 µl lysis buffer (supplied with the Dual-Light kit). Activities of β-galactosidase and luciferase were measured by the Dual-Light Chemieluminescent Reporter Gene Assay (Tropix, Bedford, MS) using a Lumat LB 9507 (EG&G Berthold, Bad Wildbad, Germany). Enzyme activities were adjusted to protein content of cell lysates, which were determinated by the BCA method (Pierce, Rockford, IL).

Quantification of GFP-positive cells

GFP, PTF–barnase and PCMV–tTA (or PNSE–tTA) cotransfected cells, subsequently cultured in the absence or presence of dox, were analysed 48 and 72 h after transfection by UV. GFP-positive cells were counted in similar sections of a number of microscopic fields. Similarly, transfected cells were rinsed from the plates with 1 ml of phosphate buffered saline (PBS) containing 0.05 mM EDTA. Numbers of GFP-positive cells were determined in a fluorescence activating cell sorter (FACS; Beckton-Dickinson) using an argon ion laser at 488 nm excitation wavelength.

Isolation of RNA and RT–PCR

Total RNAs from cultured cells were isolated by the NucleoSpin RNA II preparation method (Macherey-Nagel, Düren, Germany) with an included DNase I digest. Usually, 0.5–1 µg of total RNA in 20 µl reaction volume was subjected to reverse transcription (RT) using superscript reverse transcriptase (Life Technologies GmbH, Eggenstein, Germany). After RT, 2 µl of a 1:5 dilution was used for PCR amplification in a total volume of 20 µl, containing MasterMix-Taq (Qiagen, Hilden, Germany).

For the bacterial RNase barnase the primers were bars (5′-AGGTCTGATAATGGTCCGT-3′) and baras (5′-AAGGGAACCTGGCAGACGTC-3′); for ribosomal protein L7 mRNA, L7s (5′-AGATGTACCGCACTGAGATTC-3′) and L7as (5′-ACTTACCAAGAGACCGAGCAA-3′).

Primers were obtained from TIB MolBiol (Berlin, Germany). PCR conditions for barnase, 4 min initial denaturation at 94°C, 20–25 cycles with 1 min denaturation at 92°C, 1 min annealing at 60°C, 45 s elongation at 72°C and finally 10 min extension at 72°C; for L7 amplification, 4 min initial denaturation at 94°C, 18–22 cycles with 1 min denaturation at 94°C, 45 s annealing at 62°C, 45 s elongation at 72°C, 10 min extention at 72°C. The resulting amplication products (barnase, 210 bp; L7, 352 bp) were separated by electrophoresis on 2% agarose gels and visualised by ethidium bromide staining.

Dot blot analysis

PCR products were diluted by 20× standard saline citrat buffer (SSC), boiled and transferred via Slot blot (MinifoldRII, Schleicher & Schuell, Dassel, Germany) to nylon membranes (Pall Biodyne B, Pall, Dreieich, Germany). The DNA were fixed by baking. The blots were hybridised with [α-32P]dATP random labelled PCR amplifications of barnase and L7. After exposition (Bio-Imager, Bio-Rad, Göttingen, Germany), the signals were quantified by Bio-Rad Multi-Analyst™ version 1.1.

Documentation

Photodocumentation was done on a Zeiss Axiophot fluorescence microscope with phase contrast and UV microscopy. Images were processed by Adobe Photoshop version 6.0.

RESULTS AND DISCUSSION

Optimisation of a tc-dependent target promoter

In order to reduce the background activity of the target promoter PXTC (5), two new promoters (both of them containing multiple tet operator sites upstream of a minimal promoter) were constructed and fused to luciferase as a reporter gene. The sequences of both domains of the target promoter PXTC (5) (the minimal promoter as well as the region encoding the operators) were altered (Fig. 1). The minimal promoter, which is in the original PhCMV*-1 the region –53 to +75 of the hCMV, was exchanged against the region –45 to +1 of the plant viral cauliflower mosaic virus 35S promoter (15,19). In PTax, the ‘enhancer region’ consists of four operators and three identical spacer regions, which are different from those in PhCMV*-1. In PTF the ‘enhancer region’ was exchanged against a fragment encoding six tet operators. The difference to PTax is that the sequences between the operators are not multimerized, but each spacer encodes a different sequence. The rationale for this alteration is that weak binding sites between operator sites might lead to background activity due to synergistic effects. These promoters were tested by transient transfection assays with tTA and rtTA in different cell lines and in stable cell clones of CHO cells (Fig. 2). As control promoters, we used PXTC, the original target promoter described by Gossen and Bujard, and a mouse mammary tumor virus (MMTV) LTR derived promoter (PMtetO5), developed as tc-dependent promoter with reduced basal activity (12).

Figure 1.

Figure 1

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Target promoter constructions (PXTC, PMtetO5, PTAX, PTF) linked to luciferase or barnase used for analyses. Transcription start sites are indicated by arrows. Note that in PTF, the spacer sequences between tet operators differ from each other.

Figure 2.

Figure 2

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Comparative transactivation measurements of promoter–luciferase constructs in cell lines HeLa, HEK293, CHO, PC12 and C6 cells with either rtTA or tTA, with (+) dox (1 µg/ml) or without (–) dox. Mean values of five independent experiments 48 h after transient transfection of 100 ng target promoter construct and 500 ng PCMV-rtTA or PCMV-tTA. The mean values of promoter activity from six different CHOAA8 (PCMV-tTA) cell clones comprising low copy integrations of promoter–luciferase constructs in the uninduced state and 48 h after dox withdrawal are shown to the right.

In all cell lines tested, PTF and PMtetO5 revealed the lowest basal Luc activities in the uninduced state, which is at least 4–10-fold depending on the cell line. PTax yielded higher background activities than PTF, indicating that avoiding the multimerisation of identical spacer sequences indeed helps to reduce background activity. In the induced state, PTF reaches at least 60% of the expression levels of PXTC in cell lines HeLa, CHO, PC12 and C6. In all these cell lines, PTF activity was higher than in pMtetO5, this effect being most pronounced in HeLa cells.

In stable cell clones of CHO cells containing PCMV–tTA and a single copy integration of respective promoters, the reduction in basal activity is 12-fold with PTF whereas induction is only reduced by 25% compared to PXTC. Thus, with this simple approach, basal activity of the target promoter was drastically reduced whereas induced activities were only reduced by 50%. This resulted in an increase of the activation of gene expression upon withdrawal of tc from 16- to 140-fold. Also, in combination with rtTA, these promoters yielded lower background activities in different cell lines, which was between 8- and 12-fold.

Analysis of conditional barnase expression

The optimised tc-dependent target promoter PTF was used for conditional suicide gene expression. PTF was fused to the barnase gene to give PTF–barnase. The barnase gene contains the coding region for a B.amyloliquefaciens RNase, a small extracellular RNase protein of 110 amino acid residues, which is in vivo inhibited by barstar (14). The barnase gene has been used in transgenic plants for cell-ablation studies (20).

The constructs PTF–barnase, PCMV–tTA (ratio 1:5) and a GFP (pEGFP) vector were cotransfected and numbers of surviving cells were determined by counting GFP positive cells (Fig. 3). Usually, 48–72 h after transfection, cell numbers were reduced by 80% after tc withdrawal in all cell lines used. The observed residual cell survival might be due to the lack of simultaneous presence of all three constructs in one single cell.

Figure 3.

Figure 3

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Tetracycline-induced cell ablation by barnase in cell lines CHO, PC12, C6 and HEK with the PTF–barnase construct. Cells were cotransfected with PTF–barnase (100 ng), PCMV–tTA (500 ng) and with 100 ng pEGFP with (+) or without (–) dox; 48 h after transfection, cell numbers were determined as mean values by counting GFP+ cells in 10 independent fields of vision under the UV microscope (magnification 100×). Right diagram, for comparison, the number of HEK cells transfected as above were determined by FACS analysis.

Analysis of conditional gene expression in neuronal cells

To assess the regulated expression of barnase in neuronal cells, we generated stable Neuro2A cell clones comprising a low copy number of PTF–barnase integrates. We received barnase positive Neuro2A clones (70% of all G418 resistant cell clones) that survived the selection procedure and exerted a conditional barnase expression after transient transfection with PCMV–tTA, PCMV–rtTA, PNSE–tTA (Fig. 4), which led to cell reduction of at least 70% after 48 h. As a comparable induction rate would be achieved with a number of weak, cell-specific promoters, demonstrated by the neuronal-specific enolase (PNSE) promoter, we argue that even a low induction of the barnase gene in vivo is sufficient to ablate specific cell types in transgenic mice.

Figure 4.

Figure 4

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Tetracycline-dependent ablation of two stable barnase containing Neuro2A cell clones by PCMV–rtTA, PCMV–tTA and PNSE–tTA. Neuro2A clones A1 and D3 were transfected with 500 ng of regulator construct. 48 h after transfection either in the presence (+dox) or in the absence (–dox) of inducer, cell numbers were determined by counting 10 independent fields of vision. Cells transfected with the regulator constructs in the uninduced state (+ for tTA; – for rtTA)were taken as 100% control by definition.

To analyse the kinetics of conditional barnase expression, we generated individual CHO-AA8 (contains a stable integrate of PCMV–tTA) cell clones containing stable integrates of PTF-barnase. Out of 12 CHO cell clones positive for barnase, seven cell cones survived for a longer time period and barnase expression was tc-inducible. From this calculation, >60% of all cell clones obtained revealed conditional cell ablation by the barnase gene. A representative induction experiment with a double-stable CHO cell clone is shown in Figure 5. The mRNA levels of barnase were determined by semiquantitative RT–PCR (Fig. 5A). Induction of barnase is maximal 7 days after tc withdrawal and drops to lower levels later on (Fig. 5C). At this time point, cell morphology appeared normal whereas 9 days after tc withdrawal, a clear-cut change in cell morphology was detectable (Fig. 5B and C). and no cell showed normal morphology compared to the uninduced state, demonstrating a complete cell ablation. The reason for the delayed kinetics of Barnase accumulation or toxicity compared to maximal levels of mRNA induction is unknown. However, a similar kinetic of luciferase accumulation has been observed with this promoter in transgenic plants (19). Similar ablation results were obtained with irradiated CHO cells. These cells were also completely ablated 11 days after tc withdrawal demonstrating that even under non-proliferative conditions, Barnase can act as suicide gene (data not shown).

Figure 5.

Figure 5

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Tetracycline-dependent cell ablation in a CHO cell clone double transgenic for PCMV–tTA and PTF–barnase. (A) Increment of barnase and L7 RT–PCR products from RNA of barnase expressing CHO cells with total cycle number, quantified as Bio-Imager units. Values are given as binary logarithms. From these data, 18 cycles were chosen for subsequent experiments to ensure maximal sensitivity and linearity of the PCR reaction. (B) Time-dependent induction of barnase mRNA levels, determined by RT–PCR. Mean values of three independent samples are given as fold-induction of barnase (bar) mRNA at respective days (d) after dox withdrawal. (C and D) Morphology of a CHO cell clone with stable integrates of PTF–barnase and PCMV–tTA cultured either in the presence (+dox; C), or after withdrawal of dox (–dox; D). Cells were stained with crystal violet. Whereas cells in the presence of dox (C) show normal fibroblast morphology, only some remaining dead cells or cell debris can be seen 11 days after dox withdrawal in (D). Scale bar in (D), 5 µm. (E) Time-dependent cell survival of CHO cells at respective days after tc withdrawal. Note that the drop in cell number is delayed towards maximal barnase mRNA induction by 2 days.

We also injected the PTF–barnase construct in pronuclei to generate transgenic mice. So far, similar to the observations in cell lines, we obtained high frequencies of barnase positive offsprings: from 10 born mice, 2 founder mice aroused, which were viable and had a normal phenotype. These results imply that conditional barnase expression with PTF resulted in higher frequencies of viable littermates as observed with a PXTC–DTA construct, for which an ∼1% rate of viable offsprings (3 per 317 born offsprings; 4) was obtained.

In conclusion, we have presented a conditional suicide system based on a modified tc-dependent expression system and combined it with a prokaryotic RNase. We found that the low basal activity of PTF allows a high percentage of transgenic propagation in cell lines and mice. In combination with barnase, even low induction levels of the target promoter, usually achieved by tTA expression under cell-specific promoters, are sufficient to exert conditional cell ablation. Thus, our novel cell ablation strategy might have an impact on the molecular analysis of pathological processes in transgenic animals, in particular in the CNS.

Acknowledgments

ACKNOWLEDGEMENTS

We thank Drs M. Gossen and H. Bujard, Heidelberg, for providing plasmids pXTCLuc and pUHD 15-1, pUHD 15-2, Dr D. Spengler, Munich, for the plasmid pMtetO5-Luc. The kind gifts of cell lines by Dr H. Hauser, Braunschweig, Dr J. Cornelis, Heidelberg, C. Spilker, Magdeburg, and Dr K. Bluezstaijn, Boston, are gratefully acknowledged. We thank Dr P. Budde, Bielefeld, for help with FACS analyses. Work was supported by the VolkswagenStiftung to C.G. and J.W.B. S.L. is a fellow of the DFG, Graduate Program ‘Cellular Basics of Biotechnical Processes’. Details on the construction of plasmids are available on request.

References

  • 1.Delaney C., Brenner,M. and Messing,A. (1996) Conditional ablation of cerebellar astrocytes in postnatal transgenic mice. J. Neurosci., 16, 6908–6918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Bush T.G., Savidge,T.C., Freeman,T.C., Cox,H.J., Campbell,E.A., Mucke,L., Johnson,M.H. and Sovroniew,M.V. (1998) Fulminant Jejuno-Ileitis following ablation of enteric glia in adult transgenic mice. Cell, 93, 189–201. [DOI] [PubMed] [Google Scholar]
  • 3.Keyvani K., Baur,I. and Paulus,W. (1999) Tetracycline-controlled expression but not toxicity of an attenuated diphteria toxin mutant. Life Sci., 64, 1719–1724. [DOI] [PubMed] [Google Scholar]
  • 4.Lee P., Morley,G., Huang,Q., Fischer,A., Seiler,S., Horner,J.W., Factor,S., Vaidya,D., Jalife,J. and Fishman,G.I. (1998) Conditional lineage ablation to model human disease. Proc. Natl Acad. Sci. USA, 95, 11371–11376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Gossen M. and Bujard,H. (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promotors. Proc. Natl Acad. Sci. USA, 89, 5547–5551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Gossen M., Freundlieb,S., Bender,G., Müller,G., Hillen,W. and Bujard,H. (1995) Transcriptional activation by tetracyclines in mammalian cells. Science, 268, 1766–1769. [DOI] [PubMed] [Google Scholar]
  • 7.Furth P.A., Onge,L.St., Böger,H., Gruss,P., Gossen,M., Kistner,A., Bujard,H. and Henninghausen,L. (1994) Temporal control of gene expression in transgenic mice by a tetracycline responsive promoter. Proc. Natl Acad. Sci. USA, 91, 9302–9306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Kistner A., Gossen,M., Zimmermann,F., Jerecic,J., Ulmer,C., Lübbert,H. and Bujard,H. (1996) Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice. Proc. Natl Acad. Sci. USA, 93, 10933–10938. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Urlinger S., Baron,U., Thellmann,M., Hasan,M.T., Bujard,H. and Hillen,W. (2000) Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. Proc. Natl Acad. Sci. USA, 97, 7963–7968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Forster K., Helbl,V., Lederer,T., Urlinger,S., Wittenburg,N. and Hillen,W. (1999) Tetracycline-inducible expression systems with reduced basal activity in mammalian cells. Nucleic Acids Res., 27, 708–710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Imhof M.O., Chatellard,P. and Mermod,N. (2000) A regulatory network for the efficient control of transgene expression. J. Gene Med., 2, 107–116. [DOI] [PubMed] [Google Scholar]
  • 12.Hoffmann A., Villalba,M., Journot,L. and Spengler,D. (1997) A novel tetracycline-dependent expression vector with low basal expression and potent regulatory properties in various mammaliam cell lines. Nucleic Acids Res., 25, 1078–1079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Truss M., Bartsch,J., Schelbert,A., Haché,R.J. and Beato,M. (1995) Hormone induces binding of receptors and transcription factors to a rearranged nucleosome on the MMTV promoter in vivo. EMBO J., 14, 1737–1751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Hartley R.W. (1989) Barnase and Barstar: Two small small proteins to fold and fit together. Trends Biochem. Sci., 14, 450–454. [DOI] [PubMed] [Google Scholar]
  • 15.Böhner S., Lenk,I., Rieping,M., Herold,M. and Gatz,C. (1999) Transcriptional activator TGV mediates dexamethasone-inducible and tetracycline-inactivatable gene expression. Plant J., 19, 87–95. [DOI] [PubMed] [Google Scholar]
  • 16.Strittmatter G., Janssens,J., Opsomer,C. and Botterman,J. (1995) Inhibition of fungal disease development in plants by engineering controlled cell death. Biotechnology, 13, 1085–1089. [Google Scholar]
  • 17.Dirks W., Wirth,M. and Hauser,H. (1993) Dicistronic transcription units for gene expression in mammalian cells. Gene, 128, 247–249. [DOI] [PubMed] [Google Scholar]
  • 18.Cashman N.R., Durham,H.D., Blusztajn,J.K., Oda,K., Tabira,T., Shaw,I.T., Dahrouge,S. and Antel,J.P. (1992) Neuroblastoma x spinal cord (NSC) hybrid cell lines resemble developing motor neurons. Dev. Dyn., 194, 209–221. [DOI] [PubMed] [Google Scholar]
  • 19.Böhner S. and Gatz,C. (2001) Characterisation of novel target promoters for the dexamethasone-inducible/tetracycline-inactivatable regulator TGV using luciferase and isopentenyl transferase as sensitive reporter genes. Mol. Gen. Genet., 264, 860–870. [DOI] [PubMed] [Google Scholar]
  • 20.Beals T.P. and Goldberg,R.B. (1997). A novel cell ablation strategy blocks tobacco anther dehiscence. Plant Cell, 9, 1527–1545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Forss-Petter S., Danielson,P.E., Catsicas,S., Battenberg,E., Price,J., Nerenberg,M. and Sutcliffe,J.G. (1990) Transgenic mice expressing beta-galactosidase in mature neurons under neuron-specific enolase promoter control. Neuron, 5, 187–197. [DOI] [PubMed] [Google Scholar]

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