Purchase this article with an account.
Lili Xu, Andrew Liu, Douglas McMahon; Circadian Clock Gene Rhythms In Human Retina Muller Cells And Their Inhibition By Sirna Knockdown Of Period Genes. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4329.
Download citation file:
© 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 exhibit self-sustained clock gene rhythms in purified cultures and these rhythms were suppressed by treatment with siRNA against Per1. Here we have transfected human retinal Muller cells with Bmal1Luc and Per2Luc lentivirus vectors and have tested the roles of Per1 and Bmal1 in human Muller cell circadian rhythms using siRNA knockdown.
Purified human retina Muller cells were seeded into 6 well plates and transfected with Per2Luc or Bmal1Luc vectors when 20-30% confluent. cells. Cell selection was performed with Blasticidin S treatment for 3 days resulting in stable cell lines expressing Luciferase under the Per2 or Bmal1 promoter. The Muller cells with Per2Luc virus were serum starved overnight, trypsinized, and then incubated with siRNA (for Per1, 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 (medium 199 and L-glutamine, B27 and luciferin) and transferred to a multichannel luminometer for recording.
Human Muller cell cultures 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. Rhythms of Muller cells with Per2::Luc were suppressed for 14 cycles by treatment with siRNA against Per1 and Bmal1. Media change increased luminescence, but did not restore rhytymicity. Control siRNA and lipofectamine alone had no effect on Muller cell rhythms.
Our results show that human retinal Muller cells exhibit molecular circadian rhythms and that expression of Per1 and Bmal1 are necessary for rhythmicity in Muller cell populations. These observations could either be the result Per1 and Bmal1 siRNA inhibition of the molecular circadian rhythms in individual human Muller cells, or due to desynchronization of Muller cell populations. Future experiments using single cell imaging could differentiate between these possibilities.
This PDF is available to Subscribers Only