title: Tetracycline transcriptional silencer tightly controls transgene expression after in vivo intramuscular electrotransfer: application to interleukin 10 therapy in experimental arthritis.
abstract: The doxycycline (Dox)-inducible reverse tetracycline transactivator (rtTA) is often used to control gene expression. However, the Tet-on system displays a high background activity. To overcome this unregulated expression we used the tetracycline-dependent transcriptional silencer (tTS), which binds the tetO inducible promoter in the absence of Dox. Controlled gene expression was analyzed in vivo by delivering combinations of Dox-regulated luciferase reporter construct, rtTA, and tTS expression plasmids into mouse muscle, using electrotransfer. Elevated luciferase expression levels were observed in the absence of doxycycline, and a 10-fold induction was obtained after drug administration. In contrast, when tTS was added, background expression was dramatically lowered by three to four orders of magnitude, and induction was maintained. The tTS system was then used to control expression of a therapeutic gene in experimental arthritis. DBA/1 mice were coinjected with plasmids encoding the antiinflammatory interleukin-10 cytokine under the control of the tetO promoter, the rtTA, and the tTS. Electrotransfer resulted in a dose-dependent increase in IL-10 expression, maintained over a 3-month period, and significant inhibitory effects on collagen-induced arthritis. We conclude that the use of tTS significantly improves the utility of the rtTA system for somatic gene transfer by reducing background activity.
authors.affiliation: Genethon, CNRS URA 1923, 91000 Evry, France. perez@genethon.fr
authors.forename: Norma
authors.initials: N
authors.lastname: Perez
authors.forename: Pascale
authors.initials: P
authors.lastname: Plence
authors.forename: Virginie
authors.initials: V
authors.lastname: Millet
authors.forename: Denis
authors.initials: D
authors.lastname: Greuet
authors.forename: Caroline
authors.initials: C
authors.lastname: Minot
authors.forename: Daniele
authors.initials: D
authors.lastname: Noel
authors.forename: Olivier
authors.initials: O
authors.lastname: Danos
authors.forename: Christian
authors.initials: C
authors.lastname: Jorgensen
authors.forename: Florence
authors.initials: F
authors.lastname: Apparailly
mesh_terms: D000818:Animals; D001169:Arthritis, Experimental / Q000235:genetics* / Q000628:therapy*; D004305:Dose-Response Relationship, Drug; D018274:Electroporation; D005260:Female; D015967:Gene Expression Regulation, Viral; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000008:administration & dosage; D007273:Injections, Intramuscular; D016753:Interleukin-10 / Q000008:administration & dosage / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D010957:Plasmids; D013752:Tetracycline / Q000378:metabolism*; D014162:Transfection
chemical_list: D016753:Interleukin-10; D013752:Tetracycline