In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

Summary

This newly developed fluorescence-based technology enables long-term monitoring of the transcription of circadian clock genes in the suprachiasmatic nucleus (SCN) of freely moving mice in real-time and at a high temporal resolution.

Abstract

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene reporters. This new and easy to use in vivo fluorescence monitoring system was developed to record the transcriptional rhythm of the clock gene, Cry1, in the suprachiasmatic nucleus (SCN) of freely moving mice. To do so, a Cry1 transcription fluorescence reporter was designed and packaged into Adeno-associated virus. Purified, concentrated virus was injected into the mouse SCN followed by the insertion of an optic fiber, which was then fixed onto the surface of the brain. The animals were returned to their home cages and allowed a 1-month post-operative recovery period to ensure sufficient reporter expression. Fluorescence was then recorded in freely moving mice via an in vivo monitoring system that was constructed at our institution. For the in vivo recording system, a 488 nm laser was coupled with a 1 × 4 beam-splitter that divided the light into four laser excitation outputs of equal power. This setup enabled us to record from four animals simultaneously. Each of the emitted fluorescence signals was collected via a photomultiplier tube and a data acquisition card. In contrast to the previous bioluminescence in vivo circadian clock recording technique, this fluorescence in vivo recording system allowed the recording of circadian clock gene expression during the light cycle.

Introduction

In mammals, the suprachiasmatic nucleus (SCN) governs the whole body's circadian rhythm to coordinate an individual's response to exogenous environmental changes (e.g., light, temperature, stress, etc.)¹. Core clock components consist of Per1-3, Cry1-2, Clock, and Bmal1, and play a central role in regulating the circadian clock of each cell. Each cell in the SCN contains the transcriptional activator, CLOCK/BMAL1, which acts as a heterodimer to induce the expression of PER and CRY. The PER/CRY complex then inhibits the function of CLOCK/BMAL1 to form a transcription-translation feedb....

Protocol

All procedures in this protocol were conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) of the National Institute of Biological Sciences, Beijing, in accordance with the governmental regulations of China.

1. Construction of the Cry1 Fluorescence Reporter

Note: Previous circadian studies using the bioluminescence system^2,12,13 fuse a circadian promoter with a destabilized luciferase (dLuc) to induce rhythmic dLuc expression. To develop a fluorescence re....

Results

Fluorescence reporter design of Cry1 was show in Figure 1A. Using the approach detailed above, 500 nL of rAAV-P(Cry1)-intron336-Venus-NLS-D2 was successfully injected into the SCN of an adult mouse, and exhibited robust Venus expression (Figure 1B, 1C). Fluorescence signals recorded under 12h/12h light/dark (LD) and dark/dark (DD) conditions (Figure 2) yielded robus.......

Discussion

In contrast to ex vivo methods, such as slice culture^4,5, RT-PCR¹⁶, and in situ hybridization¹⁷, which require that animals be killed, the in vivo recording method allows investigators to study circadian gene expression in a living animal. As such, this technology provides the ability to evaluate the effect of different physical perturbations (e.g., sleep deprivation, stress, food.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
KOD Plus Neo	TOYOBO	KOD-401	Reagent
pVENUS-N1	addgene	#61854	Plasmid
pcDNA3.3_d2eGFP	addgene	#26821	Plasmid
pAAV-EF1a-double floxed-hChR2(H134R)-mCherry-WPRE-HGHpA	addgene	#20297	Plasmid
MluI	Thermo Scientific	FD0564	Reagent
EcoRI	Thermo Scientific	FD0274	Reagent
Gibson Assembly Mix	NEB	E2611s	Reagent
Lipofectamine 2000	Thermo Scientific	12566014	Reagent
Syringe Filter	EMD Millipore	SLHV033RS	0.45 µm
HiTrap heparin columns	gelifesciences	17-0406-01	1 mL
Amicon ultra-4 centrifugal filter	EMD Millipore	UFC810024	100,000 MWCO
Benzonase nuclease	Sigma-Aldrich	E1014	Reagent
Sodium deoxycholate	Sigma-Aldrich	D5670	Make fresh solution for each batch
mouse stereotaxic apparatus	B&E TEKSYSTEMS LTD	#SR-5M/6M	Equipment
pentobarbital	SigmaAldrich	#1507002	Reagent
mouse stereotaxic apparatus	B&E TEKSYSTEMS LTD	#SR-5M/6M	Equipment
Hydrogen peroxide solution	SigmaAldrich	#216763	Reagent
Optical Fiber	Thorlabs	FT200EMT	0.39 NA, Ø200 µm
microsyringe pump	Nanoliter 2000 Injector, WPI		Equipment
ceramic ferrule	Shanghai Fiblaser		230 μm I.D., 2.5 mm O.D.
Gene Observer	BiolinkOptics		Equipment