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Tetracycline Derivatives: Alternative Effectors for Tet Transregulators

2004; Future Science Ltd; Volume: 37; Issue: 4 Linguagem: Inglês

10.2144/04374bm04

1940-9818

Christel Krueger, Klaus Pfleiderer, Wolfgang Hillen, Christian Berens,

RNA Interference and Gene Delivery

BioTechniquesVol. 37, No. 4 BenchmarksOpen AccessTetracycline derivatives: alternative effectors for Tet transregulatorsChristel Krueger, Klaus Pfleiderer, Wolfgang Hillen & Christian BerensChristel KruegerFriedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany, Klaus PfleidererFriedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany, Wolfgang HillenFriedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany & Christian Berens*Address correspondence to: Christian Berens, Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstr. 5, 91058 Erlangen, Germany. e-mail: E-mail Address: cberens@biologie.uni-erlangen.deFriedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, GermanyPublished Online:6 Jun 2018https://doi.org/10.2144/04374BM04AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Conditional gene expression using the Tet system is well established in eukaryotes (1). The tetracycline-controlled transactivator (tTA) binds to Ptet−1 in the absence of an effector and stimulates transcription of the successive target gene (2). Effector tetracyclines abolish activation by tTA (Tet-Off). The reverse transactivator rtTA2S-M2 contains a mutated tetR allele and activates Ptet−1 only in the presence of an effector (Tet-On) (3).The most widely used effectors are tetracycline (tc) and doxycycline (dox) for tTA and dox for rtTA2S-M2. Several other tc derivatives have been tested for their suitability as effectors of the Tet system in tobacco and human cell lines (4–7). Here we explore the induction characteristics of eight tc derivatives for tTA and rtTA2S-M2 in HeLa cell lines. We chose the seven clinically licensed and commercially available antibiotics: tc, dox, demeclocycline (demeclo), minocycline (mino), chloro-tetracycline (Cl-tc), sancycline (san) and metacycline (meta), and additionally tigecycline (TGC), which belongs to the new group of glycylcyclines currently undergoing phase III clinical trials (www.clinicaltrials.gov). None of these derivatives, except for dox, have been tested with rtTA2S-M2 before. A structural overview is provided in Figure 1.Figure 1. Chemical structure of tetracycline (tc) analogs used in this study.The substituents are marked R5 to R9 (with respect to the corresponding C atom) and designated in the table.The HeLa cell line X1/5 contains tTA and a Ptet−1 driven luciferase reporter construct stably integrated into the genome (2,8). Cells were seeded in 24-well plates and cultivated overnight before fresh Dulbecco's modified Eagle's medium (DMEM)/10% fetal calf serum (FCS) was added with the respective effector concentration. Cells were harvested 24 h after induction. Luciferase activities and protein concentrations were determined as described (9). The expression response curves are shown in Figure 2, A and B. Activation is fully abolished at effector concentrations between 1 and 100 ng/mL. In the text, numbers given in brackets indicate effector concentrations in ng/mL at 100 standardized arbitrary light units (ALU)/µg protein. This value is in the linear phase of the logarithmic curves and was introduced to mark differences in effector efficiencies. For tTA, dox [0.04] is the most potent effector of the derivatives tested. Effectors with medium efficiency are mino [0.1] and meta [0.15] followed by san [0.25] and demeclo [0.4]. Cl-tc [2], tc [2.5], and TGC [3] are weakest. TGC had not been shown to be an inducer for TetR or its derived transregulators previously.Figure 2. Induction characteristics of tetracycline-controlled transactivator (tTA) and rtTA2S-M2 with tetracycline (tc) analogs.HeLa cells carrying the luciferase gene under Ptet−1 control and either (A and B) tTA (X1/5) or (C) rtTA2S-M2 (HR2SM2L) were grown at various effector concentrations. Luciferase activity was determined after 24 h. Values represent the means of triplicate samples with standard deviations given in arbitrary light units (ALU)/µg total cell protein. The value obtained (A and B) without effector (for tTA) or (C) with 1 µg/mL doxycycline (dox) (for rtTA2S-M2) was standardized to 1000 ALU/µg protein, and all other values were corrected accordingly. (D) HeLa cells were transiently cotransfected with 10 ng pUHrT62−1 (3) encoding rtTA2S-M2 and 100 ng pUHC13-3 (2) carrying the luciferase gene under Ptet−1 control using Lipofectamine™ (Gibco Life Technologies, Gaithersburg, MD, USA). Cells were incubated with different effector concentrations. After 24 h, luciferase activity was determined as described. Values represent the means of triplicate samples with standard deviations given in ALU/µg total cell protein corrected for transfection efficiency (9). The value obtained without effector was standardized to 1 corrected ALU/µg protein. All experiments were performed at least twice. TGC, tigecycline; demeclo, demeclocycline; mino, minocycline; Cl-tc, chlorotetracycline; san, sancycline; meta, metacycline.We then examined activation of rtTA2S-M2. The cell line HeLa X1/6 containing the Ptet−1 luciferase reporter construct (2,8) was stably transfected with PvuI linearized pWHE146 (10) to express rtTA2S-M2. HR2SM2L clones were selected with G418 and tested for inducible luciferase activity.Expression response experiments were performed as described for X1/5. As shown in Figure 2C, meta [100] has the same activation potential as the default effector dox [100]. TGC [900] is about one order of magnitude weaker. San [6000] and mino [>10,000] display noteworthy activation only if present in a concentration above 3000 ng/mL, which is, however, still obtainable in vivo (11,12). The latter three did not reach saturation within the range of effector concentrations tested. Demeclo, Cl-tc, and tc are not effectors for rtTA2S-M2 in this cell line. Thus, not every effector for tTA necessarily activates rtTA2SM2, nor does an effector's potential to activate rtTA2S-M2 reflect its effectiveness to reduce luciferase activity exerted by tTA. TGC, for example, is the weakest effector tested for tTA but an intermediate one for rtTA2S-M2. Also, meta is weaker than dox for tTA, but not for rtTA2S-M2. In general, rtTA2S-M2 responds to 10- to 100-fold higher effector concentrations than tTA.It has recently been published (7) that mino is an effector for rtTA (13), weaker than dox but with better passage of the blood-brain barrier. As mino is a poor activator for rtTA2S-M2 tested here, the more effective mino derivative TGC might be an even better alternative.We also explored activation by dox and tc in HeLa cells that were transiently cotransfected with plasmids encoding rtTA2S-M2 and the Ptet−1 driven luciferase reporter (Figure 2D). Transfection experiments were performed as described previously (9). Transiently transfected rtTA2S-M2 reacts to dox at lower concentrations than in the cell line HR2SM2L (1 ng/mL versus 10 ng/mL), and tc leads to reasonable activation at concentrations below 1000 ng/mL. Transiently and stably transfected cells differ in the amount of transactivator and reporter construct. While stable cell lines harbor only one or maximally a few copies of the respective genes, transiently transfected cells contain many. The resulting high amounts of transactivator and reporter construct support low affinity interactions and, thus, affect Tet regulation (9,10).In conclusion, activation of rtTA2S-M2 by tc analogs can be compared by dose-response analysis of HR2SM2L. However, the concentration needed to fully activate rtTA2S-M2 depends on the intracellular amounts of the system's components, which may also vary between different clones of stable cell lines. Thus, this concentration has to be determined individually for each experimental setup.Graded dox response is well known for Tet transregulators and can be exploited to achieve intermediate expression levels in cell culture (14). However, because of the high sensitivity of the transactivator for dox, the concentration window is narrow. Small variations in the dox concentration result in large differences of transcriptional output (3) (Figure 2). The higher concentrations used with less efficient derivatives might buffer pharmacokinetic variations in transgenic animals, and intermediate expression levels could be more readily maintained.So far, only dox has been used as an effector for rtTA2S-M2. Its pharmacokinetic properties render it a very good effector for most applications (5). However, there are reports about toxicity (15) and slow-release kinetics (5,6,16). Meta, an equally potent effector for rtTA2S-M2, seems to be less toxic at high concentrations (17). Its serum half-live is shorter than that of dox (18), which should lead to a more rapid switch in gene expression (16). The concentrations needed to fully induce rtTA2S-M2 are obtained in all tissues tested (11). 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This work was supported by the Deutsche Forschungsgemeinschaft through SFB473 and the Fonds der Chemischen Industrie Deutschlands. C.K. obtained a predoctoral fellowship from the State of Bavaria.Competing Interests StatementC.K., K.P., and C.B. declare no conflicts of interest. W.H. declares the development of Tet-Off and Tet-On variants.PDF download