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Kenkichi Baba, Takana Tubo, Ting-Chung Suen, Jason DeBruyne, Gianluca Tosini; Circadian Rhythm in Cone-like (661W) Photoreceptor Cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4663.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies have shown that the mammalian retina contains circadian clocks that control many physiological events with this tissue. However, the presence of circadian clocks within the photoreceptor (rods or cones) is still matter of debate. In this study, we investigate whether cone-like cells (661W, a murine photoreceptor derived cell line) contain circadian clocks and whether they express mRNA for dopamine and melatonin receptors.
661W cells generously provided by Dr. Al-Ubaidi, University of Oklahoma Health Science Center were used in this study. Cells were cultured and grown to 90-100 % confluency with Dulbecco’s Modified Eagle Medium (DMEM) at 37 oC and then were harvested and the mRNA was extracted. The presence of mRNAs for clock genes (Period1, Period2 and Bmal1), dopamine (D1-D5) and melatonin (MT1 and MT2) receptors was investigated with RT-PCR. To determine the presence of a circadian clock, 661W cells were stably transfected with a PERIOD2::LUCIFERASE (PER2::LUC) . Transfected cells (661W-PER2::LUC) were then cultured with DMEM containing 0.1mM D-Luciferin K salt, and the bioluminescence rhythms emitted from 661W-PER2::LUC cells was measured using a LumiCycle®.
RT-PCR amplification and electrophoresis gel analysis indicated that Period1, Period2, Bmal1 mRNAs were all present in 661W cells. mRNAs for the Dopamine D4 and both melatonin (MT1 and MT2) receptors was also amplifiable from these cells. 661W-PER2::LUC cells also expressed a clear circadian rhythm in bioluminescence that lasted for at least 4 days The average free running period was 21.96±0.26 hours (Mean±SEM, n=16).
Our data indicate that 661W-PER2::LUC cells contain a circadian clock and express dopamine and melatonin receptors. Thus 661W-PER2::LUC cells may represent an useful in vitro model to study the mechanisms that generate circadian rhythms and the role that melatonin and dopamine plays in the entrainment of circadian rhythm in a cone-like photoreceptor.
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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