抄録
To analyze the dynamics or persistence of clock gene expression, noninvasive real-time monitoring system using firefly luciferase is employed. Although this system enables longitudinal and quantitative monitoring of the gene expression in living cells, more than one gene expression cannot be monitored. To precisely analyze the molecular mechanism of circadian clock, it is critical to monitor multiple gene expressions and/or interactions with their transcription factors simultaneously. We have recently developed a novel in vitro reporter assay system, in which two gene expressions can be monitored simultaneously by splitting the emissions from green- and red-emitting luciferases. In this study, we have applied this system to real-time monitoring of clock gene expressions. To verify the system, promoter regions of mPer2 and mBmal1 were connected to the red and green luciferases, respectively, and the constructs were transiently co-transfected into rat1 fibroblasts. The bioluminescence emission from the cells was continuously measured with a luminometer equipped with an optical filter (ATTO Co.). The phases of Per2 and Bmal1 oscillations were antiphase, which is consistent with mRNA expression patterns. The identical result was also obtained when the luciferases were swapped. These results clearly indicate the system can accurately monitor respective gene expression profiles in the same cell populations even when these were measured simultaneously. We are now applying this system for analyzing interaction between clock or clock-controlled-gene products and response elements in clock gene promoters [Jpn J Physiol 55 Suppl:S53 (2005)]
