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.

Download full-text


Available from: Mazahir T. Hasan, Apr 19, 2014
58 Reads
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Short interfering RNAs (siRNAs) and microRNAs (miRNAs) have been widely used in mammalian tissue culture and model organisms to selectively silence genes of interest. One limitation of this technology is the lack of precise external control over the gene-silencing event. The use of photocleavable protecting groups installed on nucleobases is a promising strategy to circumvent this limitation, providing high spatial and temporal control over siRNA or miRNA activation. Here, we have designed, synthesized and site-specifically incorporated new photocaged guanosine and uridine RNA phosphoramidites into short RNA duplexes. We demonstrated the applicability of these photocaged siRNAs in the light-regulation of the expression of an exogenous green fluorescent protein reporter gene and an endogenous target gene, the mitosis motor protein, Eg5. Two different approaches were investigated with the caged RNA molecules: the light-regulation of catalytic RNA cleavage by RISC and the light-regulation of seed region recognition. The ability to regulate both functions with light enables the application of this optochemical methodology to a wide range of small regulatory RNA molecules.
    Nucleic Acids Research 09/2013; 41(22). DOI:10.1093/nar/gkt806 · 9.11 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Chemical genetics, or chemical biology, has become an increasingly powerful method for studying biological processes. The main objective of chemical genetics is the identification and use of small molecules that act directly on proteins, allowing rapid and reversible control of activity. These compounds are extremely powerful tools for researchers, particularly in biological systems that are not amenable to genetic methods. In addition, identification of small molecule interactions is an important step in the drug discovery process. Increasingly, the African frog Xenopus is being used for chemical genetic approaches. Here, we highlight the advantages of Xenopus as a first-line in vivo model for chemical screening as well as for testing reverse engineering approaches.
    genesis 03/2012; 50(3):207-18. DOI:10.1002/dvg.22009 · 2.02 Impact Factor
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The conditional expression of transgenes at high levels in sparse and specific populations of neurons is important for high-resolution optogenetic analyses of neuronal circuits. We explored two complementary methods, viral gene delivery and the iTet-Off system, to express transgenes in the brain of zebrafish. High-level gene expression in neurons was achieved by Sindbis and Rabies viruses. The Tet system produced strong and specific gene expression that could be modulated conveniently by doxycycline. Moreover, transgenic lines showed expression in distinct, sparse and stable populations of neurons that appeared to be subsets of the neurons targeted by the promoter driving the Tet-activator. The Tet system therefore provides the opportunity to generate libraries of diverse expression patterns similar to gene trap approaches or the thy-1 promoter in mice, but with the additional possibility to pre-select cell types of interest. In transgenic lines expressing channelrhodopsin-2, action potential firing could be precisely controlled by two-photon stimulation at low laser power, presumably because the expression levels of the Tet-controlled genes were high even in adults. In channelrhodopsin-2-expressing larvae, optical stimulation with a single blue LED evoked distinct swimming behaviors including backward swimming. These approaches provide new opportunities for the optogenetic dissection of neuronal circuit structure and function.
    Frontiers in Neural Circuits 12/2009; 3:21. DOI:10.3389/neuro.04.021.2009 · 3.60 Impact Factor
Show more