Synthetic rewiring and boosting type I interferon responses for visualization and counteracting viral infections.

Mammalian first line of defense against viruses is accomplished by the interferon (IFN) system. Viruses have evolved numerous mechanisms to reduce the IFN action allowing them to invade the host and/or to establish latency. We generated an IFN responsive intracellular hub by integrating the synthetic transactivator tTA into the chromosomal Mx2 locus for IFN-based activation of tTA dependent expression modules. The additional implementation of a synthetic amplifier module with positive feedback even allowed for monitoring and reacting to infections of viruses that can antagonize the IFN system. Low and transient IFN amounts are sufficient to trigger these amplifier cells. This gives rise to higher and sustained-but optionally de-activatable-expression even when the initial stimulus has faded out. Amplification of the IFN response induced by IFN suppressing viruses is sufficient to protect cells from infection. Together, this interfaced sensor/actuator system provides a toolbox for robust sensing and counteracting viral infections.

Controlled re-activation of epigenetically silenced Tet promoter-driven transgene expression by targeted demethylation.

Faithful expression of transgenes in cell cultures and mice is often challenged by locus dependent epigenetic silencing. We investigated silencing of Tet-controlled expression cassettes within the mouse ROSA26 locus. We observed pronounced DNA methylation of the Tet promoter concomitant with loss of expression in mES cells as well as in differentiated cells and transgenic animals. Strikingly, the ROSA26 promoter remains active and methylation free indicating that this silencing mechanism specifically affects the transgene, but does not spread to the host's chromosomal neighborhood. To reactivate Tet cassettes a synthetic fusion protein was constructed and expressed in silenced cells. This protein includes the enzymatic domains of ten eleven translocation methylcytosine dioxygenase 1 (TET-1) as well as the Tet repressor DNA binding domain. Expression of the synthetic fusion protein and Doxycycline treatment allowed targeted demethylation of the Tet promoter in the ROSA26 locus and in another genomic site, rescuing transgene expression in cells and transgenic mice. Thus, inducible, reversible and site-specific epigenetic modulation is a promising strategy for reactivation of silenced transgene expression, independent of the integration site.

Single-cell analysis reveals heterogeneity in onset of transgene expression from synthetic tetracycline-dependent promoters.

Synthetic promoters have been designed for mammalian cells to achieve both temporal and quantitative control over transgene expression without interfering with the endogenous cellular network. Routine applications of synthetic expression systems are based on steady-state measurements of gene expression while the mechanism by which these steady-states are realised at the single-cell level has not been investigated. We focused on the elucidation of the kinetics of doxycycline-controlled synthetic modules as a paradigm. Following gene expression in single cells, we observed a gradual increase of transgene expression within the first 48 h after activation, as determined by flow cytometry. Time-lapse microscopy revealed that the onset of transgene expression was highly variable in individual cells. Interestingly, a bidirectional cassette design showed significantly reduced cell-to-cell heterogeneity in expression. Of note, the influence of the cell cycle seems to be negligible, since the onset of expression correlates with cell division in only a minor fraction of the cell population. In contrast, rapid and synchronous transgene expression could be realized using a posttranslational regulation system that relies on ligand-induced stabilization of a tagged protein. Thus, the inherent temporal variability of transcriptionally regulated synthetic transgene expression systems has to be considered for kinetic and correlative experimental applications.