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 obtaining 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.
Muñoz
Iciar
I
Department of Fish Reproductive Physiology, Instituto de Acuicultura de Torrelasal, CSIC, Ribera de Cabanes, 12595 Torrelasal, Castellon, Spain.
Gómez
Ana
A
Zanuy
Silvia
S
Carrillo
Manuel
M
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D003001:Cloning, Molecular; D005786:Gene Expression Regulation / Q000187:drug effects; D049452:Green Fluorescent Proteins; D006801:Humans; D049409:Luciferases, Firefly; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D013752:Tetracycline / Q000494:pharmacology; D013753:Tetracycline Resistance / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics*
D000900:Anti-Bacterial Agents; D015534:Trans-Activators; D049452:Green Fluorescent Proteins; D049409:Luciferases, Firefly; D013752:Tetracycline