title: In-depth Characterization of Firefly Luciferase as a Reporter of Circadian Gene Expression in Mammalian Cells.
abstract: Firefly luciferase (Fluc) is frequently used to report circadian gene expression rhythms in mammalian cells and tissues. During longitudinal assays it is generally assumed that enzymatic substrates are in saturating excess, such that total bioluminescence is directly proportional to Fluc protein level. To test this assumption, we compared the enzyme kinetics of purified luciferase with its activity in mammalian cells. We found that Fluc activity in solution has a lower Michaelis constant (Km) for luciferin, lower temperature dependence, and lower catalytic half-life than Fluc in cells. In consequence, extracellular luciferin concentration significantly affects the apparent circadian amplitude and phase of the widely used PER2::LUC reporter in cultured fibroblasts, but not in SCN, and we suggest that this arises from differences in plasma membrane luciferin transporter activity. We found that at very high concentrations (>1 mM), luciferin lengthens circadian period, in both fibroblasts and organotypic SCN slices. We conclude that the amplitude and phase of circadian gene expression inferred from bioluminescence recordings should be treated with some caution, and we suggest that optimal luciferin concentration should be determined empirically for each luciferase reporter and cell type.
authors.affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK.
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Feeney
authors.affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK.
authors.forename: Marrit
authors.initials: M
authors.lastname: Putker
authors.affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK.
authors.forename: Marco
authors.initials: M
authors.lastname: Brancaccio
authors.affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK.
authors.forename: John S
authors.initials: JS
authors.lastname: O'Neill
mesh_terms: D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D002940:Circadian Rhythm; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D049454:Firefly Luciferin / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements / Q000379:methods; D051379:Mice; D056950:Period Circadian Proteins / Q000235:genetics / Q000378:metabolism*; D013493:Suprachiasmatic Nucleus / Q000378:metabolism; D013997:Time Factors
chemical_list: C513312:Per2 protein, mouse; D056950:Period Circadian Proteins; D049454:Firefly Luciferin; D049409:Luciferases, Firefly
keywords: bioluminescent reporter; clock gene; enzyme kinetics; firefly luciferase; mammalian circadian rhythms