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J.C. Besharse, D. Lourim, J. Fogerty, K. Freeman, H. Scoma, D. Vilceanu; Analysis of Ocular Circadian Rhythms Using Per2::Luc Knock–In Mice . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5429.
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© ARVO (1962-2015); The Authors (2016-present)
Retinas of Xenopus laevis exhibit high amplitude circadian rhythms, and their photoreceptors are the site of an endogenous circadian oscillator. Recent studies of clock gene expression in rodents, however, have suggested significant differences in their retinal circadian organization. To better clarify circadian organization in the mouse eye we used Per2::luc knock–in mice to monitor circadian rhythms in ocular tissues in vitro, and correlated rhythmic luciferase activity with the expression of endogenous Per2 using in situ hybridization (ISH) and immunofluorescence (IFL).
Ocular tissues from Per2::luc knock–in mice were cultured in darkness for multiple days under sensitive photomultiplier tubes in HEPES buffered MEM supplemented with luciferin (Yoo, et al., 2004, PNAS. 101: 5339). Endogenous clock gene expression was monitored using quantitative ISH with 33P labeled probes and by IFL.
Rhythms of luciferase activity were routinely monitored for 4 to 7 days in Per2::luc explants of RPE–choroid, cornea, and iris, but were not detected in neural retina or lens from the same eyes. Phasing of Per2::luc rhythms were different between cornea and RPE–choroid. Quantitative ISH revealed robust rhythms of endogenous Per2 in the non–pigmented layer of the ciliary body, the RPE–choroid, and photoreceptors; phasing in the ciliary body and RPE–choroid were similar. Both quantitative ISH and IFL revealed either near constitutive (non–rhythmic) expression or extremely low amplitude rhythms of endogenous Per2 in the inner nuclear and ganglion cell layers; levels of expression in these layers exceeded that in photoreceptors by at least 2–fold. Micro–dissections of iris and cornea that included all or portions of the ciliary body exhibited particularly robust Per2::luc rhythms.
We have detected robust rhythms of Per2 in photoreceptors, RPE–choroid, and ciliary body using quantitative ISH and in the RPE–choroid and cornea using the Per2::luc mice. Retinal Per2::luc rhythms may be obscured by several factors including the high level of near–constitutive Per2 expression in the inner retina, and or insufficiency of culture conditions. Differences in phasing of the ocular rhythms suggest differences in the mechanism of phase control.
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