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Lili Xu, Tianyu Xu, Guoxiang Ruan, Douglas G. McMahon; Inhibition Of Muller Cell Circadian Rhythms By siRNA Knockdown Of Period Genes. Invest. Ophthalmol. Vis. Sci. 2011;52(14):915.
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The mammalian retina contains a self-sustained circadian clock, but the precise roles of retinal cell types and clock genes in retinal rhythms remain to be defined. We have shown in previously that retinal Muller cells exhibit self-sustained clock gene rhythms in purified cultures and that the clock gene Period1 (Per1), is necessary for overall retinal molecular circadian rhythms. Here we have tested the role of Per1 in Muller cell circadian rhythms using siRNA knockdown.
Purified Muller cell cultures were derived from PERIOD2::Luciferase (PER2::LUC) transgenic mice and propagated for 3-5 passages. Muller cells were serum starved overnight, trypsinized, and then incubated with mixture of siRNA (for Per1, Per2, control siRNA) and Lipofectamine for 25min at room temperature. Cells were then seeded onto 35mm dishes contained serum free of DMEM and incubated at 37°C in 5% CO2 and 95% ambient air for 6 hours, switched to 10% FBS DMEM were for 6 hours then changed into recording medium (medium 199 and L-glutamine, B27 and luciferin) and transferred to a multichannel luminometer for recording.
Muller cell cultures exhibited robust free-running rhythms in PER2::LUC bioluminescence that were maintained for at least 6 cycles following a media change. These rhythms were suppressed for 6 cycles by treatment with siRNA against Per1 and then could be recovered upon subsequent media change. As a positive control for the effectiveness of suppression, siRNA against Per2 suppressed luminescence output of the PER2::LUC reporter. Control siRNA and lipofectamine alone had no effect on Muller cell rhythms.
Our results show that Muller cells exhibit molecular circadian rhythms and that expression of Per1 is necessary for rhythmicity in Muller cell populations. These observations could either be the result Per1 siRNA inhibition of the molecular circadian rhythms in individual Muller cells, or due to desynchronization of Muller cell populations. Future experiments using single cell imaging could differentiate between these possibilities.
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