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Nucleic Acids Research 2005 33(16):e137; doi:10.1093/nar/gni137
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Published online 7 September 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

Methods Online

Stringent doxycycline-dependent control of gene activities using an episomal one-vector system

Georg W. Bornkamm*, Christian Berens1, Conny Kuklik-Roos, Jean-Marie Bechet2, Gerhard Laux, Jürgen Bachl, Martin Korndoerfer, Martin Schlee, Michael Hölzel, Anastassia Malamoussi, Rob D. Chapman, Falk Nimmerjahn3, Josef Mautner3, Wolfgang Hillen1, Hermann Bujard4 and Jean Feuillard5

GSF-Institut für Klinische Molekularbiologie und Tumorgenetik Marchioninistrasse 25, D-81377 München, Germany 1Lehrstuhl für Mikrobiologie der Universität Erlangen-Nürnberg Staudtstrasse 5, D-91058 Erlangen, Germany 2Guest scientist, UMR 6551 Université de Caen F-14074 Caen, France 3Klinische Kooperationsgruppe, Pädiatrische Tumorimmunologie, Kinderklinik der Technischen Universität München Germany 4Zentrum für Molekulare Biologie der Universität Heidelberg Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany 5UMR CNRS 6101, Faculté de Médecine de Limoges et Laboratoire d'Hématologie, Centre Hospitalier Universitaire Dupuytren 2, Avenue Martin Luther King, F-87042 Limoges, France

*To whom correspondence should be addressed. Tel: +49 89 7099510; Fax: +49 89 7099500; Email: bornkamm{at}gsf.de

Received July 21, 2005. Revised August 19, 2005. Accepted August 19, 2005.

Conditional expression systems are of pivotal importance for the dissection of complex biological phenomena. Here, we describe a novel EBV-derived episomally replicating plasmid (pRTS-1) that carries all the elements for conditional expression of a gene of interest via Tet regulation. The vector is characterized by (i) low background activity, (ii) high inducibility in the presence of doxycycline (Dox) and (iii) graded response to increasing concentrations of the inducer. The chicken beta actin promoter and an element of the murine immunoglobin heavy chain intron enhancer drive constitutive expression of a bicistronic expression cassette that encodes the highly Dox-sensitive reverse tetracycline controlled transactivator rtTA2S-M2 and a Tet repressor-KRAB fusion protein (tTSKRAB) (silencer) placed downstream of an internal ribosomal entry site. The gene of interest is expressed from the bidirectional promoter Ptetbi-1 that allows simultaneous expression of two genes, of which one may be used as surrogate marker for the expression of the gene of interest. Tight down regulation is achieved through binding of the silencer tTSKRAB to Ptetbi-1 in the absence of Dox. Addition of Dox releases repression and via binding of rtTA2S-M2 activates Ptetbi-1.

Present address: Falk Nimmerjahn, Department of Immunology and Autoimmune Diseases, Rockefeller University, NY, USA


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