Figure 1

Construction of a gene network for regulated transgene expression in vivo. (A) The regulatory network-based expression system assessed in this study consists of 3 plasmid components encoding a constitutively expressed repressor, an inducible activator, and a reporter or therapeutic gene regulated by both the repressor and the activator proteins. Without doxycycline, the repressor protein binds to and represses the promoters of the two other plasmids. Doxycycline binds to and inactivates the repressor, which relieves the activator promoter from repression and allows low expression levels to establish. As the activator is expressed, it activates it own expression as well as that of the reporter gene, gradually increasing expression to maximally induced levels. (B) The strength of various repressor constructs was evaluated by the electrotransfer of the network’s three plasmid components into the tibialis anterior muscle of wild type mice, using the luciferase coding sequence as a reporter gene. Mice were provided doxycycline (+ dox) or not (- dox) in the drinking water and they were sacrificed after two weeks. The relative luciferase activity was determined from muscle extracts. Numbers indicate the fold-induction of normalized luciferase activities by doxycycline. (C) The strength of the prokaryotic part of the chimeric TetR-TR450W repressor was compared when expressing the fusion protein either from the bacterial DNA sequence of from a DNA sequence with “humanized” codons [24]. (D) Performance of the network system controlled by the humanized TetR (hTetR) fused to the TR450W repressor domain expressed from the β actin promoter in mice muscles, as a comparison to that obtained from the original pTet-ON™ system (Clontech) electrotransferred similarly. Fold activations obtained upon the addition of doxycyclin are as indicated above each filled bar. Each column represents the mean of values obtained from 16 muscles of 8 mice per condition.