Evaluation of the tetracycline- and ecdysone-inducible systems for expression of neurotransmitter receptors in mammalian cells.
Establishing a stable cell line that expresses a particular protein of interest is often a laborious and time-consuming experience. With constitutive expression systems, a gradual loss of the highly expressing clones over a given time span and/or a severe counter-selection due to toxicity of the expressed protein for the host cell line are major drawbacks. In both cases, inducible expression systems offer a valuable alternative. Over the years, many regulated expression systems have been developed and evaluated. In the present study, we compare the efficiency, the advantages and the drawbacks of a tetracycline- and an ecdysone-inducible system for expression of the reporter protein chloramphenicol acetyltransferase and of different G-protein-coupled serotonin (5-HT) receptors. A high level of expression of different 5-HT receptors was obtained with the tetracycline-inducible system. In the cell line L929, which stably expresses the tetracycline-responsive transactivator, a maximum ligand binding of 20,000 and 9500 fmol/mg protein was measured for the h5-HT(1B) and h5-ht(1F) receptors, respectively. In the HEK293rtTA cell line, levels of 15,700, 3000, and 9100 fmol bound ligand/mg protein were obtained for the h5-HT(1B), h5-ht(1F) and h5-HT(4b) receptors, respectively. These high expression levels remained stable for several months of continuous culture. Although the ecdysone-inducible expression system was useful for tightly regulated expression, the levels were far lower than those obtained with the tetracycline system (e.g. 640 fmol bound ligand/mg protein for the h5-ht(1F) receptor in HEK293EcR).
Van Craenenbroeck
K
K
Department of Molecular Biology, University of Gent and VIB, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium.
Vanhoenacker
P
P
Leysen
J E
JE
Haegeman
G
G
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D004440:Ecdysone / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000096:biosynthesis; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D051379:Mice; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D044302:Receptor, Serotonin, 5-HT1B; D017981:Receptors, Neurotransmitter / Q000096:biosynthesis*; D011985:Receptors, Serotonin / Q000096:biosynthesis; D044422:Receptors, Serotonin, 5-HT4; D013752:Tetracycline / Q000494:pharmacology*; D014162:Transfection; D000096642:Receptor, Serotonin, 5-HT1F
D000900:Anti-Bacterial Agents; D015500:Chloramphenicol O-Acetyltransferase; D004440:Ecdysone; D019204:GTP-Binding Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009419:Nerve Tissue Proteins; D044302:Receptor, Serotonin, 5-HT1B; D017981:Receptors, Neurotransmitter; D011985:Receptors, Serotonin; D044422:Receptors, Serotonin, 5-HT4; D013752:Tetracycline; D000096642:Receptor, Serotonin, 5-HT1F