A one-step approach to obtain cell clones expressing tetracycline- responsive transactivators

Despite the wide application of the tetracycline-regulated gene expression system, several drawbacks in establishing the system in in vitro-cultured cells have been described. Most of the problems are related to the obtainment of a reliable tetracycline-regulated cell clone, which often results in arduous labor. We describe here a new approach to facilitate the screening and selection of such cell clones. We have constructed a tetracycline-responsive plasmid that harbors an antibiotic resistance gene fused to the enhanced green fluorescent protein (EGFP) gene and the luciferase gene, both under the control of a bidirectional promoter. We demonstrate that the selection of tetracycline-regulated clones is highly simplified by using this plasmid. Only clones expressing the system in a functional manner are able to survive under antibiotic selection. In addition, a quick characterization of the responsiveness of the clones is possible by monitoring GFP expression in vivo.This work was supported by the EC Grant FAIR CT98-3482 and the MCYT Grant MAR-1999-1647-CE. I.M. was supported by a fellowship from Diputación de Castellón