Methods for Detecting PER2:LUCIFERASE Bioluminescence Rhythms in Freely Moving Mice.
J Biol Rhythms
; 37(1): 78-93, 2022 02.
Article
em En
| MEDLINE
| ID: mdl-34873943
ABSTRACT
Circadian rhythms are driven by daily oscillations of gene expression. An important tool for studying cellular and tissue circadian rhythms is the use of a gene reporter, such as bioluminescence from the reporter gene luciferase controlled by a rhythmically expressed gene of interest. Here we describe methods that allow measurement of circadian bioluminescence from a freely moving mouse housed in a standard cage. Using a LumiCycle In Vivo (Actimetrics), we determined conditions that allow detection of circadian rhythms of bioluminescence from the PER2 reporter, PER2LUC, in freely behaving mice. The LumiCycle In Vivo applies a background subtraction that corrects for effects of room temperature on photomultiplier tube (PMT) output. We tested delivery of d-luciferin via a subcutaneous minipump and in the drinking water. We demonstrate spikes in bioluminescence associated with drinking bouts. Further, we demonstrate that a synthetic luciferase substrate, CycLuc1, can support circadian rhythms of bioluminescence, even when delivered at a lower concentration than d-luciferin, and can support longer-term studies. A small difference in phase of the PER2LUC bioluminescence rhythms, with females phase leading males, can be detected with this technique. We share our analysis scripts and suggestions for further improvements in this method. This approach will be straightforward to apply to mice with tissue-specific reporters, allowing insights into responses of specific peripheral clocks to perturbations such as environmental or pharmacological manipulations.
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Base de dados:
MEDLINE
Assunto principal:
Ritmo Circadiano
/
Proteínas Circadianas Period
Limite:
Animals
Idioma:
En
Revista:
J Biol Rhythms
Assunto da revista:
FISIOLOGIA
Ano de publicação:
2022
Tipo de documento:
Article