In-depth characterization of firefly luciferase as a reporter of circadian gene expression in mammalian cells
Author(s)
Feeney, Kevin A
Putker, Marrit
Brancaccio, Marco
O'Neill, John S
Type
Journal Article
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.
Date Issued
2016-12-01
Date Acceptance
2016-10-01
Citation
Journal of Biological Rhythms, 2016, 31 (6), pp.540-550
ISSN
0748-7304
Publisher
SAGE Publications
Start Page
540
End Page
550
Journal / Book Title
Journal of Biological Rhythms
Volume
31
Issue
6
Copyright Statement
© 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 3.0 License (http://www.creativecommons.org/licenses/by/3.0/) which permits any use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access page (https://us.sagepub.com/en-us/nam/open-access-at-sage).
Identifier
http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000387721500002&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=1ba7043ffcc86c417c072aa74d649202
Subjects
Science & Technology
Life Sciences & Biomedicine
Biology
Physiology
Life Sciences & Biomedicine - Other Topics
firefly luciferase
bioluminescent reporter
clock gene
mammalian circadian rhythms
enzyme kinetics
CATALYZED-REACTIONS
BIOLUMINESCENCE
CLOCK
REVEALS
RHYTHMS
MOUSE
TIME
PHOSPHORYLATION
MUTANTS
BIOLOGY
Publication Status
Published
Date Publish Online
2016-10-10