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

Autor(es)

Gabriel, Gabriele V. M. ^{[1, 2, 3]} ; Viviani, Vadim R. ^{[1, 2, 3]}

Afiliação do(s) autor(es)

^[1] Fed Univ Sao Carlos UFSCar, Grad Sch Evolut Genet & Mol Biol, Sao Carlos, SP - Brazil

^[2] Fed Univ Sao Carlos UFScar, Grad Sch Biotechnol & Environm Monitoring, Sorocaba, SP - Brazil

^[3] Fed Univ Sao Carlos UFSCar, Dept Phys Chem & Math, Lab Biochem & Biotechnol Bioluminescence, Sorocaba, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES; v. 13, n. 12, p. 1661-1670, 2014.
• Citações Web of Science: 9

Resumo

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. (AU)

• Processo FAPESP: 11/23961-0 - Luciferases pH-sensitivas: origem estrutural da sensibilidade ao pH, e viabilidade de emprego em biossensores celulares de pH e cátions de metais pesados
• Beneficiário: Vadim Viviani
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 14/04477-9 - Desenvolvimento de biossensores raciométricos bioluminescentes de pH e metais divalentes baseados na engenharia da região sensível ao pH nas luciferases de vagalumes
• Beneficiário: Gabriele Verônica de Mello Gabriel
• Modalidade de apoio: Bolsas no Brasil - Doutorado