Spatially-restricted transgene expression in vivo by two-photon release of small molecule inducers – LocaGenEx

The patterns of neuronal network activity are fundamental to brain function. To correlate changes in network activity with genetic manipulation of neurons, we want to establish a method that allows targeted transgene expression in single cells in vivo by irradiation with two-photon light so that the manipulated neuron can be analysed before, during, and after gene expression. To photoactivate transgene expression with high spatio-temporal control, a method based on the inducible Tetracycline (Tet) system and the reversible inhibition of tetracycline analogs with photoremovable protecting groups (“caging”) will be used. Previously, photoactivated gene expression has been successfully demonstrated in vitro. However, in vivo applications are experimentally much more challenging and thus it is crucial to optimize a few important photo- and biophysical properties of the photoactivatable tetracycline analogs. Therefore, we plan to develop a total synthesis approach for the flexible production of caged tetracycline analogs with high two-photon sensitivity. This synthetic approach will allow us to efficiently modify analogs and quickly adjust their properties according to the experimental needs in vivo. The goal is to photoactivate Kir2.1, a gene that cell-autonomously silences the electrical activity of neurons, in a subset of cells and to investigate its influence on neuronal network activity of larger ensembles. Network activity will be visualized by calcium imaging and the network patterns will be analyzed before and after Kir2.1 expression. Ultimately, in vivo photoactivated gene expression using two-photon exciation will prove to be a very powerful tool for biological research in general and for neurobiology and developmental biology in particular.