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Improved single-chain transactivators of the Tet-On gene expression system

Xue Zhou1, Jori Symons1, Rieuwert Hoppes1, Christel Krueger2, Christian Berens2, Wolfgang Hillen2, Ben Berkhout1 and Atze T Das1*

Author Affiliations

1 Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands

2 Lehrstuhl fur Mikrobiologie, Institut fur Mikrobiologie, Biochemie und Genetik, Friedrich-Alexander-Universitat Erlangen-Nurnberg, Staudtstrasse 5, D-91058 Erlangen, Germany

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BMC Biotechnology 2007, 7:6 doi:10.1186/1472-6750-7-6

Published: 19 January 2007

Abstract

Background

The Tet-Off (tTA) and Tet-On (rtTA) regulatory systems are widely applied to control gene expression in eukaryotes. Both systems are based on the Tet repressor (TetR) from transposon Tn10, a dimeric DNA-binding protein that binds to specific operator sequences (tetO). To allow the independent regulation of multiple genes, novel Tet systems are being developed that respond to different effectors and bind to different tetO sites. To prevent heterodimerization when multiple Tet systems are expressed in the same cell, single-chain variants of the transactivators have been constructed. Unfortunately, the activity of the single-chain rtTA (sc-rtTA) is reduced when compared with the regular rtTA, which might limit its application.

Results

We recently identified amino acid substitutions in rtTA that greatly improved the transcriptional activity and doxycycline-sensitivity of the protein. To test whether we can similarly improve other TetR-based gene regulation systems, we introduced these mutations into tTA and sc-rtTA. Whereas none of the tested mutations improved tTA activity, they did significantly enhance sc-rtTA activity. We thus generated a novel sc-rtTA variant that is almost as active and dox-sensitive as the regular dimeric rtTA. This variant was also less sensitive to interference by co-expressed TetR-based tTS repressor protein and may therefore be more suitable for applications where multiple TetR-based regulatory systems are used.

Conclusion

We developed an improved sc-rtTA variant that may replace regular rtTA in applications where multiple TetR-based regulatory systems are used.