Novel application of pH-sensitive firefly luciferases as dual reporter genes for simultaneous ratiometric analysis of intracellular pH and gene expression/location

Photochemical & Photobiological Sciences - 2014
Gabriele V. M. Gabriel1,2,3, Vadim R. Viviani2,3,1
1Laboratory of Biochemistry and Biotechnology of Bioluminescence, Department of Physics, Chemistry and Mathematics, Federal University of São Carlos (UFSCar), Sorocaba, Brazil
2Graduate School of Evolutive Genetics and Molecular Biology, Federal University of São Carlos (UFSCar), São Carlos, Brazil
3Graduate School of Biotechnology and Environmental Monitoring, Federal University of São Carlos (UFSCar), Sorocaba, Brazil

Tóm tắt

Firefly luciferases are widely used as bioluminescent reporter genes for bioimaging and biosensors. Aiming at simultaneous analyses of different gene expression and cellular events, luciferases and GFPs that exhibit distinct bioluminescence and fluorescence colors have been coupled with each promoter, making dual and multicolor reporter systems. Despite their wide use, firefly luciferase bioluminescence spectra are pH-sensitive, resulting in a typical large red shift at acidic pH, a side-effect that may affect some bioanalytical purposes. Although some intracellular pH-indicators employ dual color and fluorescent dyes, none has been considered to benefit from the characteristic spectral pH-sensitivity of firefly luciferases to monitor intracellular pH-associated stress, an important indicator of cell homeostasis. Here we demonstrate a linear relationship between the ratio of intensities in the green and red regions of the bioluminescence spectra and pH using firefly luciferases cloned in our laboratory (Macrolampis sp2 and Cratomorphus distinctus), allowing estimation of E. coli intracellular pH, thus providing a new analytical method for ratiometric intracellular pH-sensing. This is the first dual reporter system that employs a single luciferase gene to simultaneously monitor intracellular pH using spectral changes, and gene expression and/or ATP concentration using the bioluminescence intensity, showing great potential for real time bioanalysis of intracellular processes associated with metabolic changes such as apoptosis, cell death, inflammation and tissue acidification, among the other physiological changes.

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