Stringent doxycycline-dependent control of gene activities using an episomal one-vector system.
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.
Bornkamm
Georg W
GW
GSF-Institut für Klinische Molekularbiologie und Tumorgenetik, Marchioninistrasse 25, D-81377 München, Germany. bornkamm@gsf.de
Berens
Christian
C
Kuklik-Roos
Conny
C
Bechet
Jean-Marie
JM
Laux
Gerhard
G
Bachl
Jürgen
J
Korndoerfer
Martin
M
Schlee
Martin
M
Hölzel
Michael
M
Malamoussi
Anastassia
A
Chapman
Rob D
RD
Nimmerjahn
Falk
F
Mautner
Josef
J
Hillen
Wolfgang
W
Bujard
Hermann
H
Feuillard
Jean
J
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
D049408:Luminescent Agents; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008156:Luciferases; D013752:Tetracycline; D004318:Doxycycline