The cumate gene-switch: a system for regulated expression in mammalian cells
1 Institut de Recherche en Biotechnologie, Conseil National de Recherches du Canada, 6100 Royalmount Avenue, Montréal, Québec, H4P 2R2, Canada
2 INRS-IAF, Université du Québec, Laval, Québec, H7N 4Z3, Canada
3 Départment de microbiologie et immunologie de l'Université de Montréal, Montréal, Québec, H3C 3J7, Canada
4 Canada's Michael Smith Genome Sciences Centre, BC Cancer Agency, 570 West 7th Avenue, Vancouver, BC, V5Z 4S6, Canada
5 Invitrogen, 688 East Main Street, Branford, CT, 06405, USA
6 AstraZeneca, 7171, Frédérick-Banting, Ville St.-Laurent, Montréal, Québec, H4S 1Z9, Canada
BMC Biotechnology 2006, 6:43 doi:10.1186/1472-6750-6-43Published: 3 November 2006
A number of expression systems have been developed where transgene expression can be regulated. They all have specific characteristics making them more suitable for certain applications than for others. Since some applications require the regulation of several genes, there is a need for a variety of independent yet compatible systems.
We have used the regulatory mechanisms of bacterial operons (cmt and cym) to regulate gene expression in mammalian cells using three different strategies. In the repressor configuration, regulation is mediated by the binding of the repressor (CymR) to the operator site (CuO), placed downstream of a strong constitutive promoter. Addition of cumate, a small molecule, relieves the repression. In the transactivator configuration, a chimaeric transactivator (cTA) protein, formed by the fusion of CymR with the activation domain of VP16, is able to activate transcription when bound to multiple copies of CuO, placed upstream of the CMV minimal promoter. Cumate addition abrogates DNA binding and therefore transactivation by cTA. Finally, an adenoviral library of cTA mutants was screened to identify a reverse cumate activator (rcTA), which activates transcription in the presence rather than the absence of cumate.
We report the generation of a new versatile inducible expression system.