Targeted modification of the Per2 clock gene alters circadian function in mPer2luciferase (mPer2Luc) mice.

Martin R Ralph,Martin Sládek,Shu-Qun Shi,Priya Crosby,Alena Sumová,Adam R Stinchcombe,Pavel Houdek,Carl H Johnson,Tenjin C Shrestha,John S O’Neill,Christopher S Colwell

doi:10.1371/journal.pcbi.1008987

Martin R Ralph, Martin Sládek + Show 9 more

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https://doi.org/10.1371/journal.pcbi.1008987

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Abstract

Modification of the Per2 clock gene in mPer2Luc reporter mice significantly alters circadian function. Behavioral period in constant dark is lengthened, and dissociates into two distinct components in constant light. Rhythms exhibit increased bimodality, enhanced phase resetting to light pulses, and altered entrainment to scheduled feeding. Mechanistic mathematical modelling predicts that enhanced protein interactions with the modified mPER2 C-terminus, combined with differential clock regulation among SCN subregions, can account for effects on circadian behavior via increased Per2 transcript and protein stability. PER2::LUC produces greater suppression of CLOCK:BMAL1 E-box activity than PER2. mPer2Luc carries a 72 bp deletion in exon 23 of Per2, and retains a neomycin resistance cassette that affects rhythm amplitude but not period. The results show that mPer2Luc acts as a circadian clock mutation illustrating a need for detailed assessment of potential impacts of c-terminal tags in genetically modified animal models.

Highlights

A commonly used and highly efficient approach for monitoring cellular processes is to engineer genes encoding critical components of the process, so that they create benign products which signal accurately the natural activity of that gene
A widely used circadian reporter expresses firefly luciferase regulated by the clock gene Period 2 (Per2) [1]
The mPER2::LUC protein is mPER2 fused with a large LUC protein attached to its C-terminus, a region whose integrity is crucial for the stability of the protein and its interaction with CRY1/2 required for both CRY and PER longevity and nuclear translocation of PER2 [42, 43], and its central role in the translation feedback loop (TTFL) of the molecular clock

Summary

Introduction

A commonly used and highly efficient approach for monitoring cellular processes is to engineer genes encoding critical components of the process, so that they create benign products which signal accurately the natural activity of that gene. An ideal reporter should reproduce the phase, amplitude, and period of the organism’s biological clock without affecting the operation of the timing mechanism itself. A widely used circadian reporter (mPer2Luc) expresses firefly luciferase regulated by the clock gene Period 2 (Per2) [1]. The engineered gene encodes a fusion protein (PER2::LUCIFERASE, PER2::LUC) which in the presence of the substrate, luciferin, produces rhythms of bioluminescence. The signal is considered to be a high-fidelity reflection of newly translated PER2 in recordings from organotypic tissue culture, freely moving animals, and cultured cells [1,2,3,4,5]

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