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Lili Xu, Andrew Liu, John S Penn, Douglas McMahon; Inhibition of Muller Cell Circadian Rhythms by siRNA Knockdown of Period Genes. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4193. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
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 mouse retinal Muller cells (MMC) of C57BL/6J exhibit self-sustained clock gene rhythms and that Per1, is necessary for overall retinal molecular circadian rhythms. Here we have tested the clock gene rhythms in MMC of C3fh+/+ and MT1-/- and MT1,2-/- and the role of Per1and Bmal1 in MMC circadian rhythms using siRNA knockdown.
Purified C3fh, MT1-/- and MT1,2-/- MMC were seeded into 6 well plates and transfected with Per2Luc or Bmal1Luc vectors. Cell selection was performed with Blasticidin S treatment resulting in stable cell lines expressing Luciferase. C3fh+/+/Per2Luc MMC were serum starved overnight, trypsinized, and then incubated with siRNA (for Per1, Per2, Bmal1, control siRNA) and Lipofectamine for 25min at room temperature. Cells were then seeded onto 35mm dishes containing serum free of DMEM and incubated at 37°C in 5% CO2 and 95% ambient air for 6 hours, switched to 10% FBS DMEM for 6 hours, then changed into recording medium and transferred to a multichannel luminometer for recording
C3fh+/+ and MT1-/- MMC exhibited robust free-running rhythms in PER2::LUC and BMAL1::LUC bioluminescence that were maintained for at least 6 cycles and were partially restores in amplitude following a media change. MT12-/- MMC exhibited the rhythms in BMAL1::LUC bioluminescence and disappeared in PER2::LUC bioluminescence. Rhythms of C3fh+/+/Per2::Luc MMC were suppressed for 14 cycles by treatment with siRNA against Per1, Per2 and Bmal1. Media change increased luminescence, but did not restore rhythmicity. Control siRNA and lipofectamine alone had no effect on MMCrhythms.
Our results show that C3fh+/+ and MT1-/- MMC exhibit molecular circadian rhythms in PER2::LUC and BMAL1::LUC bioluminescence. MT12-/- MMC exhibit the rhythms in BMAL1::LUC bioluminescence. The expression of Per1 and Bmal1 are necessary for rhythmicity in C3fh+/+ MMC populations. These observations could either be the result Per1 and Bmal1 siRNA inhibition of the molecular circadian rhythms in individual C3fh MMC, or due to desynchronization of MMC populations. Future experiments using siRNA transfection in MT1-/- and MT12-/- MMC could differentiate between these possibilities.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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