Nishide Shin-ya, Honma Sato, Nakajima Yoshihiro, Ikeda Masaaki, Baba Kenkichi, Ohmiya Yoshihiro, Honma Ken-ichi
Department of Physiology, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan.
Genes Cells. 2006 Oct;11(10):1173-82. doi: 10.1111/j.1365-2443.2006.01015.x.
A new reporter system for monitoring expressions of two clock genes, Per1 and Bmal1, from a single tissue in culture was developed in mice. Reporters are Vargula hilgendorfii luciferase (VL) and firefly luciferase (FL), whose activities are increased in parallel with Per1 and Bmal1 expressions, respectively. Formal properties of the circadian system in transgenic mice are indistinguishable from those in wild-type animals. Circadian rhythms in Per1-VL and Bmal1-FL in the suprachiasmatic nucleus (SCN) were robust and anti-phasic, although they were phase delayed by 4-8 h as compared with circadian rhythms in respective transcript levels in vivo. In peripheral tissues such as liver, circadian rhythms in Bmal1-FL persisted for more than 3 weeks. In the course of prolonged culture, circadian rhythms apparently damped out, but were restored immediately by refreshment of the culture medium. Restoration of the circadian rhythm is unlikely to be due to resetting of desynchronized population oscillation, because peripheral circadian rhythms did not show a type 0 phase response curve (PRC) for medium refreshment, a requirement for instantaneous resetting of circadian oscillation. Long-term persistence of circadian oscillation in spite of external perturbations supports an idea that circadian oscillations in peripheral tissues are self-sustained.
在小鼠中开发了一种新的报告系统，用于监测来自培养的单一组织中的两个生物钟基因Per1和Bmal1的表达。报告基因是海萤荧光素酶（VL）和萤火虫荧光素酶（FL），它们的活性分别与Per1和Bmal1的表达平行增加。转基因小鼠中昼夜节律系统的形式特性与野生型动物中的无法区分。视交叉上核（SCN）中Per1-VL和Bmal1-FL的昼夜节律很强且呈反相，尽管与体内各自转录水平的昼夜节律相比，它们的相位延迟了4-8小时。在肝脏等外周组织中，Bmal1-FL的昼夜节律持续超过3周。在长时间培养过程中，昼夜节律明显减弱，但通过更换培养基可立即恢复。昼夜节律的恢复不太可能是由于不同步群体振荡的重置，因为外周昼夜节律对培养基更换未显示出0型相位响应曲线（PRC），而这是昼夜振荡瞬时重置的必要条件。尽管存在外部干扰，昼夜振荡仍能长期持续，这支持了外周组织中的昼夜振荡是自我维持的观点。
