title: Stringent doxycycline-dependent control of gene activities using an episomal one-vector system.
abstract: 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 rtTA2(S)-M2 and a Tet repressor-KRAB fusion protein (tTS(KRAB)) (silencer) placed downstream of an internal ribosomal entry site. The gene of interest is expressed from the bidirectional promoter P(tet)bi-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 tTS(KRAB) to P(tet)bi-1 in the absence of Dox. Addition of Dox releases repression and via binding of rtTA2(S)-M2 activates P(tet)bi-1.
authors.affiliation: GSF-Institut für Klinische Molekularbiologie und Tumorgenetik, Marchioninistrasse 25, D-81377 München, Germany. bornkamm@gsf.de
authors.forename: Georg W
authors.initials: GW
authors.lastname: Bornkamm
authors.forename: Christian
authors.initials: C
authors.lastname: Berens
authors.forename: Conny
authors.initials: C
authors.lastname: Kuklik-Roos
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Bechet
authors.forename: Gerhard
authors.initials: G
authors.lastname: Laux
authors.forename: Jürgen
authors.initials: J
authors.lastname: Bachl
authors.forename: Martin
authors.initials: M
authors.lastname: Korndoerfer
authors.forename: Martin
authors.initials: M
authors.lastname: Schlee
authors.forename: Michael
authors.initials: M
authors.lastname: Hölzel
authors.forename: Anastassia
authors.initials: A
authors.lastname: Malamoussi
authors.forename: Rob D
authors.initials: RD
authors.lastname: Chapman
authors.forename: Falk
authors.initials: F
authors.lastname: Nimmerjahn
authors.forename: Josef
authors.initials: J
authors.lastname: Mautner
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Hillen
authors.forename: Hermann
authors.initials: H
authors.lastname: Bujard
authors.forename: Jean
authors.initials: J
authors.lastname: Feuillard
mesh_terms: D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004318:Doxycycline / Q000494:pharmacology*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000032:analysis; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D008156:Luciferases / Q000032:analysis; D049408:Luminescent Agents / Q000032:analysis; D008213:Lymphocyte Activation; D010957:Plasmids; D051381:Rats; D013601:T-Lymphocytes / Q000276:immunology; D013752:Tetracycline / Q000494:pharmacology
chemical_list: D049408:Luminescent Agents; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008156:Luciferases; D013752:Tetracycline; D004318:Doxycycline