Doxycycline-dependent photoactivated gene expression in eukaryotic systems

Max Planck Institute of Neurobiology, Munich-Martinsried, Germany.
Nature Methods (Impact Factor: 32.07). 07/2009; 6(7):527-31. DOI: 10.1038/nmeth.1340
Source: PubMed


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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Available from: Mazahir T. Hasan, Apr 19, 2014
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    • "A brief irradiation with non-damaging UV light removes the caging group and restores activity of the biomolecule. This methodology has been successfully applied to the light-regulation of gene expression through caged antisense agents (9–14), caged mRNA (15), caged DNA decoys (16), caged triplex-forming oligonucleotides (17), caged proteins (18–20), caged small molecules (21,22) and recently to caged miRNA antagomirs (23,24). "
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    • "To provide a light-sensitive transcriptional control system, Cambridge et al. recently designed two caged analogues of tetracycline: caged doxycyline and caged cyanodoxycline (Cambridge et al., 2009). The second compound has the additional advantage of reduced membrane permeability, to increase perdurance within the cell after uncaging. "
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    • "The Tet system provides several advantages over other conditional gene expression systems in zebrafish (Esengil et al., 2007; Emelyanov and Parinov, 2008) including (1) minimal side effects on gene regulation (Reboredo et al., 2008), (2) no detectable toxicity of the modulator (Dox), (3) no interference with hormone signaling, and (4) the availability of optimized components that have been tested extensively in other species (Gossen and Bujard, 1992; Baron et al., 1997; Tang et al., 2009). Moreover, the recent development of caged Dox provides the opportunity to fine-tune the control of gene expression using optical approaches (Cambridge et al., 2009). "
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