Controlled transgene expression by E1-E4-defective adenovirus vectors harbouring a "tet-on" switch system.

BACKGROUND

The "tet switch system" was originally described under the tet-off configuration with its components encoded by two separate plasmids. Since then, many virus vectors harbouring tet-off components have been designed and their regulation by tetracycline is widely reported. On the contrary, tet-on regulation by viral vectors is poorly documented.

METHODS

E1-E4-defective adenoviruses harbouring either rtTA or the luciferase gene under a minimal inducible promoter (TK* or CMV*) or both components in a single genome were produced. Using either a double or a single virus strategy, induction of luciferase expression was investigated in various cell lines, in mice muscle and in rat brain.

RESULTS

Over 400-fold induction can be reached with PC12 and NHA cells using a double virus strategy. Comparison of the background activity of different minimal inducible promoters revealed a significant difference between TK* and CMV* promoters both with the cell culture and the in vivo experiments. Interestingly, a single virus strategy permitted an induction exceeding 600-fold with human astrocyte primary cells. Moreover, the E1-E4-defective adenovirus-mediated tet-on system can be quickly switched off and turned back on again. Depending on the cell line, the level of rtTA derived by the single virus strategy differed, resulting in different efficiencies. Experiments performed in rat striatum and mouse muscle confirmed the importance of rtTA expression and minimal promoter used on both doxycycline-independent expression and induction efficiency. Under appropriated rtTA expression, a 32-fold induction is observed in mouse muscle.

CONCLUSIONS

In the recombinant adenovirus context, the CMV* but not the TK* promoter is sensitive to transcriptional interference resulting in high doxycycline-independent expression. By paying attention to the rtTA expression, moderate and high induction can be obtained in vivo and in vitro accordingly.