Tetracycline transcriptional silencer (tTS) tightly controls transgene expression in the skeletal muscle: in vivo intramuscular IL-10 DNA electrotransfer application to arthritis

Background

The Dox-inducible reverse tetracycline transactivator (rtTA) is often used to control gene activity in cells and transgenic animals. However, the Tet-on system is limited by the high levels of transgene expression in the absence of doxycycline administration.

Objective and methods

To overcome this unregulated expression, we used the tetracycline-dependent transcriptional silencer (tTS) that binds the tetO inducible promoter in the absence of Dox. Controlled gene expression was analyzed in vivo by delivering combinations of the Dox-regulated luciferase reporter construct, the rtTA and the tTS expression plasmids into the tibialis anterior muscles of adult mice, using electrotransfer.

Results

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. The tibialis anterior muscle of DBA1 mice was coinjected with plasmids encoding the anti-inflammatory cytokine viral interleukin-10 (vIL-10) under the control of the tetO promoter, the rtTA and tTS. Electroporation of injected muscles resulted in a dose-dependent increase in vIL-10 expression, maintained over a 3-month period. The Dox-regulated vIL-10 secretion showed significant inhibitory effects on collagen-induced arthritis, as paw swelling was reduced and the onset of disease was delayed in the Dox-treated group in comparison with the control group without Dox.

Conclusion

The use of tTS significantly improves the utility of the rtTA system for somatic gene transfer by reducing background activity.