Tetracycline derivatives: alternative effectors for Tet transregulators.

BENCHMARKS Conditional gene expression using the Tet system is well established in eukaryotes (1). The tetracycline-controlled transactivator (tTA) binds to P tet-1 in the absence of an effector and stimulates transcription of the successive target gene (2). Effector tet-racyclines abolish activation by tTA (Tet-Off). The reverse transactivator rtTA2 S-M2 contains a mutated tetR al-lele and activates P tet-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 rtTA2 S-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 rtTA2 S-M2 in HeLa cell lines. We chose the seven clinically licensed and commercially available antibiotics: tc, dox, demeclocycline (demeclo), mi-nocycline (mino), chloro-tetracycline (Cl-tc), sancycline (san) and metacy-cline (meta), and additionally tigecy-cline (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 rtTA2 S-M2 before. A structural overview is provided in Figure 1. The HeLa cell line X1/5 contains tTA and a P tet-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 ef-fector 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 efficien-cies. 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. We then examined activation of rtTA2 S-M2. The cell line HeLa X1/6 containing the P tet-1 luciferase reporter construct (2,8) was stably transfected …