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Heba Khader Victor Solodushko Abu Bakr Al-Mehdi Jonathon Audia Brian Fouty 《Journal of fluorescence》2014,24(2):305-311
Tetracycline-inducible systems allow for either suppression or induction of transgene expression to facilitate studies of cell physiology. Doxycycline is a preferred inducer for these gene expression systems due to its membrane permeability; however, the heterocyclic structure of doxycycline exhibits fluorogenic properties that can potentially bias measurement of other fluorochromes. Thus the simultaneous use of tetracycline-inducible systems and fluorescent proteins as reporter genes or as intracellular biosensors may lead to potentially confounding results. Herein, using cells which co-express the ratiometric redox sensitive intracellular reporter, roGFP, and a tetracycline-inducible reporter plasmid encoding the reporter gene, mCherry, as a model system, we describe the overlapping intracellular fluorescent signals between doxycycline and commonly used intracellular fluorescent probes. In our cells, the addition of doxycycline to cells caused a dose- and time-dependent increase in cell fluorescence with 405 nm excitation which overlapped with that of the oxidized configuration of roGFP. Incubating cells in concentrations of doxycycline less than 1 μg/mL and removing doxycycline from the media 60 min before performing experiments eliminated fluorescence interference while still maintaining maximal reporter transgene activation. 相似文献
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Shivani Garapaty-Rao Christopher Nasveschuk Alexandre Gagnon Eric Y. Chan Peter Sandy Jennifer Busby Srividya Balasubramanian Robert Campbell Feng Zhao Louise Bergeron James E. Audia Brian K. Albrecht Jean-Christophe Harmange Richard Cummings Patrick Trojer 《Chemistry & biology》2013,20(11):1329-1339
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