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Kenkichi Baba, Ting-Chung Suen, Aida Sanchez-Bretano, Jason DeBruyne, P. Michael Iuvone, Gianluca Tosini; Removal of the clock gene Bmal1 from 661W cells affects cell viability and response to oxidative stress. Invest. Ophthalmol. Vis. Sci. 2018;59(9):969.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies have indicated that the removal of Bmal1, a key component of the molecular circadian clock in mammals, from the retina alters visual sensitivity and cone photoreceptors development in mice. Additional work has also shown that the removal of Bmal1 from neural retina may affect the viability of cone photoreceptors during aging. We have previously shown that 661W cells (a cone photoreceptor-like cell line) contain a functional circadian clock. The aim of the present study was to generate 661W cells in which the circadian clock was disabled by removal of Bmal1 and to investigate whether Bmal1 removal affects their viability and response to an oxidative stress challenge.
Bmal1 knock-out 661W cells (661W-BKO) were generated from Per2-luc 661W cell using CRISPR/Cas9 gene editing. Deletion of Bmal1 from 661W was verified with western blot using BMAL1 antibody and by monitoring Per2-luc bioluminescence circadian rhythms using a Lumicycle. To investigate cell viability in 661W, the number of cells in the cultured dish was counted over the course of 10 days using the cell counter. To investigate the effect of Bmal1 removal on an oxidative stress challenge, 661W and 661W-KO cells were treated with hydrogen peroxide (H2O2,1mM) at two time points for two hours (subjective day and night) and then cell viability was assessed.
We successfully generated several lines in which BMAL1 was no longer detectable and no circadian rhythm in Per2-luc bioluminescence was observed. Removal of Bmal1 reduced the growth rate of 661W cells. 661W-BKO viability after the oxidative stress challenge was significantly reduced with respect to 661W cells. Interestingly, the survival rate to an oxidative stress challenge in 661W cell showed a circadian response with higher survival during the subjective night. No circadian variation in the response to an oxidative stress challenge was observed in 661W-BKO cells.
We successfully generated in vitro model for the circadian disruption in a cone-like photoreceptor cell line. Our results indicate that removal of Bmal1 from the 661W cells altered the cell growth and sensitivity to the oxidative stress. Thus, 661W-BKO cells may represent a good model to study the mechanisms by which the circadian clock may affect the photoreceptor response to oxidative stress and cell viability.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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