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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. Author summary: Engineered genes that are modified to express bioluminescent signals can be used to monitor cellular processes by reflecting the actual state of the process in real time. For circadian rhythm studies, a widely used reporter model (mPer2Luc) expresses firefly luciferase under the control of the clock gene Period 2 (Per2), encoding a fusion protein (PER2::LUCIFERASE) which produces rhythms of bioluminescence. We present evidence that the engineered protein produces extensive changes in iconic circadian behavioral patterns in mice. The evidence comes from analysis of long-term recordings of circadian behavior in mPer2Luc mice in constant darkness and constant light, together with responsiveness to light pulses and entrainment to restricted food availability. By integrating a well-established mathematical model of circadian rhythm generation in the suprachiasmatic nuclei with the known functional heterogeneity within the nucleus, we recapitulated the observed locomotor behavioral patterns in WT and mPer2Luc mice. Functional alterations to the stability of PER2 protein predicted from the model were substantiated by increased repression strength of PER2::LUC over WT PER2 on CLOCK:BMAL1 transcriptional activation. Therefore, mPer2Luc is a clock mutation that provides new insights into the functional neuroanatomy of the mammalian circadian system and the critical importance of Per2 regulation in rhythm generation. [ABSTRACT FROM AUTHOR]