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Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling
Published on: June 25, 2015
Graded or threshold response of the tet-controlled gene expression: all depends on the concentration of the
Niels Heinz1, Katharina Hennig, Rainer Loew
1EUFETS GmbH, Idar-Oberstein, 55743, Germany.
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.
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