Development of Novel Circadian Reporter

Abstract

Adaptations to light and dark cycles on Earth are integral to the survival of all organisms. Indeed, all living organisms retain some form of biological rhythms that ebb and flow with daily environmental stimuli. Importantly, disruptions to circadian rhythms (CRs) are associated with reduction in life/health span and numerous metabolic disorders. As such, there is substantial interest in regulating CRs to improve health outcomes. In mammals, these CRs are generated by a transcriptional-translational feedback loop composed of core clock proteins BMAL1, CLOCK, PERIOD and CRY. Other proteins include RORa and REV-ERBa, which regulate expression of BMAL1 and CLOCK. Alongside light stimuli, CRs are sensitive to environmental cues such as food intake. There has been recent interest in the role that presence of key nutrients, specifically lipid species, plays in regulating CRs. However, the mechanism by which these metabolites act upon the circadian clock is unknown. One limitation of CR-centered research is that it is inherently low-throughput. CR studies typically incur a large financial cost and require large amounts of labor to process and analyze biological samples due to the sheer number of time points and biological replicates. As such, the field would benefit from an accurate circadian reporter that is sensitive to changes to clock gene expression. Here, we present a novel circadian reporter that can streamline circadian rhythm data collection and analysis with the use of PERIOD2-promoter driven fluorescent reporter. We tested the efficacy of the reporter in AML12 cells stably expressing it and treated them with several known circadian inducers to determine complete and robust circadian profiles. In future directions, we aim to optimize experimental design parameters to enhance performance of the novel circadian reporter.