In-depth Characterization of Firefly Luciferase as a Reporter of Circadian Gene Expression in Mammalian Cells
Open Access
- 10 October 2016
- journal article
- research article
- Published by SAGE Publications in Journal of Biological Rhythms
- Vol. 31 (6), 540-550
- https://doi.org/10.1177/0748730416668898
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.Keywords
This publication has 46 references indexed in Scilit:
- Firefly luciferase: an adenylate-forming enzyme for multicatalytic functionsCellular and Molecular Life Sciences, 2009
- CKIε/δ-dependent phosphorylation is a temperature-insensitive, period-determining process in the mammalian circadian clockProceedings of the National Academy of Sciences of the United States of America, 2009
- At Least Four Distinct Circadian Regulatory Mechanisms Are Required for All Phases of Rhythms in mRNA AmountJournal of Biological Rhythms, 2008
- Pyrophosphate and tripolyphosphate affect firefly luciferase luminescence because they act as substrates and not as allosteric effectorsThe FEBS Journal, 2008
- Kinetic analysis and modeling of firefly luciferase as a quantitative reporter gene in live mammalian cellsBiotechnology & Bioengineering, 2004
- Oxygen regulation and limitation to cellular respiration in mouse skeletal muscle in vivoAmerican Journal of Physiology-Heart and Circulatory Physiology, 2003
- Mammalian TOR: A Homeostatic ATP SensorScience, 2001
- Site-Directed Mutagenesis of Firefly Luciferase Active Site Amino Acids: A Proposed Model for Bioluminescence ColorBiochemistry, 1999
- Circadian Clock Mutants in Arabidopsis Identified by Luciferase ImagingScience, 1995
- Firefly LuciferaseAdvances in enzymology and related areas of molecular biology, 1976