Evaluation of the tetracycline- and ecdysone-inducible systems for expression of neurotransmitter receptors in mammalian cells

Authors


: Dr Guy Haegeman, as above.
E-mail: Guy.Haegeman@dmb.rug.ac.be

Abstract

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-HT1B and h5-ht1F receptors, respectively. In the HEK293rtTA cell line, levels of 15 700, 3000, and 9100 fmol bound ligand/mg protein were obtained for the h5-HT1B, h5-ht1F and h5-HT4b 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-ht1F receptor in HEK293EcR).

Ancillary