In-depth Characterization of Firefly Luciferase as a Reporter of Circadian Gene Expression in Mammalian Cells

Feeney, Kevin A.; Putker, Marrit; Brancaccio, Marco; O’Neill, John S.

doi:10.1177/0748730416668898

Cited by 42 publications

(31 citation statements)

References 48 publications

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“…In the peroxyoxalate ester systems, light emission of bis(6-alkoxycarbonyl-2,4-dichlorophenyl) oxalates could last more than 12 h with low-intensity 16 . In firefly BL, typical emission could last for more than 6 h when firefly luciferase was in live cells and 2 h when firefly luciferase was in solution 17 , 18 . The light emission of our CL system was even visible to naked eyes and lasted for over 150 h. Accordingly, our system have distinguished CL intensity and duration time, which superior to non-enzymatic peroxyoxalate ester CL system and enzymatic firefly BL system.…”

Section: Resultsmentioning

confidence: 99%

Firefly-mimicking intensive and long-lasting chemiluminescence hydrogels

Liu

Shen

et al. 2017

Nat Commun

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Most known chemiluminescence (CL) reactions exhibit flash-type light emission. Great efforts have been devoted to the development of CL systems that emit light with high intensity and long-lasting time. However, a long-lasting CL system that can last for hundreds of hours is yet-to-be-demonstrated. Here we show firefly-mimicking intensive and long-lasting CL hydrogels consisting of chitosan, CL reagent N-(4-aminobutyl)-N-ethylisoluminol (ABEI) and catalyst Co2+. The light emission is even visible to naked eyes and lasts for over 150 h when the hydrogels are mixed with H2O2. This is attributed to slow-diffusion-controlled heterogeneous catalysis. Co2+ located at the skeleton of the hydrogels as an active site catalyzes the decomposition of slowly diffusing H2O2, followed by the reaction with ABEI to generate intensive and long-lasting CL. This mimics firefly bioluminescence system in terms of intensity, duration time and catalytic characteristic, which is of potential applications in cold light sources, bioassays, biosensors and biological imaging.

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Section: Resultsmentioning

confidence: 99%

Firefly-mimicking intensive and long-lasting chemiluminescence hydrogels

Liu

Shen

et al. 2017

Nat Commun

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“…Notably, if a direct correlation of GFP to Fluc expression were needed, a measurement of the actual protein levels and the catalytic active protein, respectively, would be necessary. 34 The T2A linker regulates the bicistronic expression and not protein stability. Furthermore, spectral unmixing should be tested with other improved pairs of luciferases and luciferins such as AkaLumine-HCl 8 with enhanced sensitivity for deep tissue imaging.…”

Section: Discussionmentioning

confidence: 99%

Quantitative in vivo dual-color bioluminescence imaging in the mouse brain

et al. 2019

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Bioluminescence imaging (BLI) is an optical imaging method that can be translated from the cell culture dish in vitro to cell tracking in small animal models in vivo. In contrast to the more widely used fluorescence imaging, which requires light excitation, in BLI the light is exclusively generated by the enzyme luciferase. The luciferase gene can be engineered to target and monitor almost every cell and biological process quantitatively in vitro and even from deep tissue in vivo. While initially used for tumor imaging, bioluminescence was recently optimized for mouse brain imaging of neural cells and monitoring of viability or differentiation of grafted stem cells. Here, we describe the use of bright color-shifted firefly luciferases (Flucs) based on the thermostable x5 Fluc that emit red and green for effective and quantitative unmixing of two human cell populations in vitro and after transplantation into the mouse brain in vivo. Spectral unmixing predicts the ratio of luciferases in vitro and a mixture of cells precisely for cortical grafts, however, with less accuracy for striatal grafts. This dual-color approach enables the simultaneous visualization and quantification of two cell populations on the whole brain scale, with particular relevance for translational studies of neurological disorders providing information on stem cell survival and differentiation in one imaging session in vivo.

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“…Substrate availability has been shown to influence circadian parameters in luciferase reporter assays (Feeney et al , ). To ensure that the effects we observed with DHEA were robust and not due to insufficient substrate, we tested different concentrations of luciferin.…”

Section: Methodsmentioning

confidence: 99%

Identification of circadian clock modulators from existing drugs

et al. 2018

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Chronic circadian disruption due to shift work or frequent travel across time zones leads to jet‐lag and an increased risk of diabetes, cardiovascular disease, and cancer. The development of new pharmaceuticals to treat circadian disorders, however, is costly and hugely time‐consuming. We therefore performed a high‐throughput chemical screen of existing drugs for circadian clock modulators in human U2OS cells, with the aim of repurposing known bioactive compounds. Approximately 5% of the drugs screened altered circadian period, including the period‐shortening compound dehydroepiandrosterone (DHEA; also known as prasterone). DHEA is one of the most abundant circulating steroid hormones in humans and is available as a dietary supplement in the USA. Dietary administration of DHEA to mice shortened free‐running circadian period and accelerated re‐entrainment to advanced light–dark (LD) cycles, thereby reducing jet‐lag. Our drug screen also revealed the involvement of tyrosine kinases, ABL1 and ABL2, and the BCR serine/threonine kinase in regulating circadian period. Thus, drug repurposing is a useful approach to identify new circadian clock modulators and potential therapies for circadian disorders.

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In-depth Characterization of Firefly Luciferase as a Reporter of Circadian Gene Expression in Mammalian Cells

Cited by 42 publications

References 48 publications

Firefly-mimicking intensive and long-lasting chemiluminescence hydrogels

Firefly-mimicking intensive and long-lasting chemiluminescence hydrogels

Quantitative in vivo dual-color bioluminescence imaging in the mouse brain

Identification of circadian clock modulators from existing drugs

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