Developing Mammalian Cellular Clock Models Using Firefly Luciferase Reporter.

Abstract

In mammals, many aspects of metabolic, physiological, and behavioral processes are regulated by endogenous circadian clocks. Oscillators of different tissue types share a common molecular mechanism at the cellular and molecular level which underlies the rhythmic expression of genes. Individual cells are the functional units for rhythm generation and cell-based clock models offer experimental tractability for discovery. Cellular clock models can be developed by introducing a noninvasive and readily detectable luciferase bioluminescence reporter as a rhythmic output, in which the promoter of a rhythmically expressed gene is fused with the firefly luciferase (Luc) gene. The bioluminescence expression in the cells is measured continuously over several days using a highly sensitive and automated recording device. As such, the data are of high temporal resolution and allow precise determination of key circadian parameters including period length, amplitude, damping rate, and phase. Miniaturization of the assays improves throughput for large scale screens. In our lab, we have expertise for constructing circadian reporters and developing reporter cell lines. Here, we describe the procedure for establishing a stable mouse hepatocyte reporter cell line. The procedure described here can be applied to various other cell types.