Luciferase activity entrainment of In vitro circadian rhythms

Abstract

An organism’s reaction to the day and night cycle, also known as its circadian rhythm, has long been studied to better understand biology. A transgenic luciferase reporting systems is one method used to study these circadian rhythms. This reporting system allows for the monitoring of genes involved in circadian rhythm which are frequently expressed in a continuously oscillating manner. These oscillations can be influenced by a variety of environmental stimuli. Interestingly, data from the Kikyo lab suggests luciferase activity itself is an overlooked synchronizer of circadian rhythms in vitro. The Kikyo lab hypothesizes a byproduct of luciferase activity is responsible for this influence on the circadian rhythm.There were two specific aims to this study. The first aim was to determine if any region of the Per2 promoter was involved in this luciferase activity driven synchronization. To this end, various pCDH-Per2::Luc plasmid constructs with differing Per2 promoter lengths were constructed for the eventual transduction into cells. Two of three targeted plasmid constructs were assembled and are currently available for transduction. The second aim was to determine if the luciferase activity byproduct P-AMPK was responsible for synchronizing the circadian rhythms of in vitro cells. To determine if P-AMPK was responsible for synchronizing cells, growth and expansion of primary Per2::Luc mesenchymal stem cells (MSCs) to be used in a variety of assays was required. However, I had difficulties culturing these MSCs and was therefore unable to carry out many of the assays needed. Nevertheless, bioluminescence recordings of these primary Per2::Luc MSCs indicated luciferase activity synchronizes the circadian rhythm of these in vitro cells. The documented culturing issues, along with the already constructed plasmids, will likely aid future researchers in the Kikyo lab to determine the cause of this phenomenon.