Background

Almost all living organisms exhibit physiological and behavioral circadian rhythms that are driven by the circadian clock (Takahashi, 2017). The circadian clock enables maximum expression of genes at appropriate times of the day, allowing organisms to appropriately adapt to environmental rhythms generated by the Earth’s rotation. The clockwork consists of ubiquitous, cell-autonomous and clock gene-driven negative feedback loops of transcription (Schibler et al., 2015). In mammals, the transcription factors BMAL1 and CLOCK activate the transcription of clock and clock-related genes such as Period (Per) and Cryptochrome (Cry) via E-box elements. PER, together with CRY, a potent transcriptional inhibitor, subsequently function to negatively regulate this complex (Kume et al., 1999).

Ex vivo tissue-culture experiments are often performed in the field of circadian biology (Yamazaki et al., 2000). The major aim of these experiments is to evaluate the circadian characteristics of clock gene expression such as period length at the tissue-autonomous level and to compare these characteristics with those at the whole-body level (Liu et al., 2007). For example, the effect of the dysfunction of a clock gene in question can be investigated by comparing the effects in ex vivo tissue culture and behavior and physiology. This culture method is also used to examine the tissue specificity of circadian entrainment factors (Sato et al., 2014). Specifically, this technique can be used to reveal in which tissue a humoral factor in question modulates circadian phase or amplitude. Additionally, although controversial, some studies suggest that the circadian phase observed in ex vivo cultured tissues can be used to estimate that in vivo (Stokkan et al., 2001).

In the present study, we developed an experimental method to analogize and evaluate circadian characteristics based on ex vivo culture of mouse whisker follicles. Briefly, individual whisker follicles are carefully dissected from mice carrying a luciferase gene whose expression is driven by a circadian promoter, and bioluminescence is measured in real time using a photomultiplier tube. This method can be useful for a wide range of applications as mentioned above.