Abstract

Background

Currently, the step-wise integration of tet-dependent transactivator and tet-responsive expression unit is considered to be the most promising tool to achieve stable tet-controlled gene expression in cell populations. However, disadvantages of this strategy for integration into primary cells led us to develop an “All-In-One” vector system, enabling simultaneous integration of both components. The effect on tet-controlled gene expression was analyzed for retroviral “All-In-One” vectors expressing the M2-transactivator either under control of a constitutive or a new type of autoregulated promoter.

Results

Determination of luciferase activity in transduced cell populations indicated improvement of the dynamic range of gene expression for the autoregulated system. Further differences were observed regarding induction kinetics and dose–response. Most notably, introduction of the autoregulated system resulted in a threshold mode of induction, whereas the constitutive system exhibited pronounced effector-dose dependence.

Conclusion

Tet-regulated gene expression in the applied autoregulated system resembles a threshold mode, whereby full induction of the tet-unit can be achieved at otherwise limiting doxycycline concentrations.

Keywords:

Tet-controlled gene expression; Transactivator concentration; Threshold response; Self-contained; Autoregulated