Abstract
High spatial and temporal resolution of conditional gene expression is typically difficult to achieve in whole tissues or organisms. We synthesized two reversibly inhibited, photoactivatable ('caged') doxycycline derivatives with different membrane permeabilities for precise spatial and temporal light-controlled activation of transgenes based on the 'Tet-on' system. After incubation with caged doxycycline or caged cyanodoxycycline, we induced gene expression by local irradiation with UV light or by two-photon uncaging in diverse biological systems, including mouse organotypic brain cultures, developing mouse embryos and Xenopus laevis tadpoles. The amount of UV light needed for induction was harmless as we detected no signs of toxicity. This method allows high-resolution conditional transgene expression at different spatial scales, ranging from single cells to entire complex organisms.
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Acknowledgements
We thank I. Mansuy and O. Griesbeck for critically reading the manuscript, D. Brown and members of his laboratory for help with the photoactivation of the Xenopus tadpoles, V. Staiger for conducting the electrophysiological measurements, C. Huber and F. Voss for technical assistance, and P. Schmieder and M. Beerbaum for recording the NMR spectra. This work was supported by the Max Planck Society. Additional support came from the Ford Foundation, the Minorities in Neuroscience Fellowship Program and a grant of the Volkswagen-Stiftung to S.B.C., F.C. and W.B.H were supported by the Federal Ministry of Education and Research (BMBF) in the framework of the National Genome Research Network, Systematic Methodological Platform RNAi, Förderkennzeichen 5 (NGFN-2).
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Cambridge, S., Geissler, D., Calegari, F. et al. Doxycycline-dependent photoactivated gene expression in eukaryotic systems. Nat Methods 6, 527–531 (2009). https://doi.org/10.1038/nmeth.1340
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DOI: https://doi.org/10.1038/nmeth.1340
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