Recording of Diurnal Gene Expression in Peripheral Organs of Mice Using the RT-Biolumicorder

Abstract

There is high interest in investigating the daily dynamics of gene expression in mammalian organs, for example in liver. Such studies help to elucidate how and with what kinetics peripheral clocks integrate circadian signals from the suprachiasmatic nucleus, which harbors the circadian master pacemaker, with other systemic and environmental cues, such as those associated with feeding and hormones. Organ sampling around the clock, followed by the analysis of RNA and/or proteins, is the most commonly used procedure in assessing rhythmic gene expression.However, this method requires large cohorts of animals and is only applicable to behaviorally rhythmic animals whose phases are known. Real-time recording of gene expression rhythms using luciferase reporters has emerged as a powerful method to acquire continuous, highresolution datasets from freely moving individual mice. Here, we share our experience and protocols with this technique, using the RT-Biolumicorder setup.

A conditional Smg6 mutant mouse model reveals circadian clock regulation through the nonsense-mediated mRNA decay pathway

“…Thus, we expected that the long-period mPER2::LUC rhythms seen in liver explants ex vivo would lead to a change in phase in vivo. To evaluate this prediction, we used a method for the real-time recording of daily liver gene expression in freely moving mice ( 31 , 32 ) that relies on luciferase reporters, luciferin delivery via an osmotic minipump, and highly sensitive bioluminescence photon counting ( Fig. 3E ).…”

“…Adult male mice, 12 to 20 weeks of age, carrying the genetically encoded circadian reporter allele mPer2::Luc ( 28 ) were used for the RT-Biolumicorder experiments. The experimental procedure followed our recently published protocol ( 31 ). Briefly, ALZET mini-osmotic pumps (model 100D5 or 2001) were filled with d -luciferin sodium salt (90 mg/ml), dissolved in PBS (pH 7.4) under sterile conditions.…”

A novel Smg6 mouse model reveals regulation of circadian period and daily CRY2 accumulation through the nonsense-mediated mRNA decay pathway

“…Thus, we expected that the long period mPER2-Luc rhythms seen in liver explants ex vivo would lead to a change in phase in vivo. To evaluate this prediction, we used a method for the realtime recording of daily liver gene expression in freely moving mice 29,30 that relies on luciferase reporters, luciferin delivery via an osmotic minipump, and highly sensitive bioluminescence photon counting (Fig. 2F).…”

“…Prior implantation the dorsal area of the mouse at the site where the liver is positioned was shaved using an electric razor. The RT-Biolumicorder (Lesa-Technology) consists of a cylindrical cage for a single mouse with photon-reflecting walls, equipped with a photomultiplier tube (PMT), water and food containers and a built-in infrared sensor that records locomotor activity ( 29,30 ). The RT-Biolumicorder records photon and activity levels in 1 min intervals.…”

“…The RT-Biolumicorder records photon and activity levels in 1 min intervals. The data, which also contain light and food access information, were saved as text files and later analyzed using the MatLab-based "Osiris" software according to 29 or a custom-made R script.…”