Wu Yue, Tian Tian, Wu Yin, Yang Yu, Zhang Yunfei, Qin Ximing
Department of Health Sciences, Institutes of Physical Science and Information Technology, Anhui University, Hefei 230601, China.
Moeden Experiment Technology Center, Institutes of Physical Science and Information Technology, Anhui University, Hefei 230601, China.
Biology (Basel). 2021 Nov 18;10(11):1204. doi: 10.3390/biology10111204.
Mammalian circadian genes are capable of producing a self-sustained, autonomous oscillation whose period is around 24 h. One of the major characteristics of the circadian clock is temperature compensation. However, the mechanism underlying temperature compensation remains elusive. Previous studies indicate that a single clock gene may determine the temperature compensation in several model organisms. In order to understand the influence of each individual clock gene on the temperature compensation, twenty-three well-known mammalian clock genes plus and genes were knocked out individually, using a powerful gene-editing tool, CRISPR/Cas9. First, , , and were knocked out as examples to verify that deleting genes by CRISPR is effective and precise. Cell lines targeting twenty-two genes were successfully edited in mouse fibroblast NIH3T3 cells, and off-target analysis indicated these genes were correctly knocked out. Through measuring the luciferase reporters, the circadian periods of each cell line were recorded under two different temperatures, 32.5 °C and 37 °C. The temperature compensation coefficient Q was subsequently calculated for each cell line. Estimations of the Q of these cell lines showed that none of the individual cell lines can adversely affect the temperature compensation. Cells with a longer period at lower temperature tend to have a shorter period at higher temperature, while cells with a shorter period at lower temperature tend to be longer at higher temperature. Thus, the temperature compensation is a fundamental property to keep cellular homeostasis. We further conclude that the temperature compensation is a complex gene regulation system instead of being regulated by any single gene. We also estimated the proliferation rates of these cell lines. After systematically comparing the proliferation rates and circadian periods, we found that the cell growth rate is not dependent on the circadian period.
哺乳动物的昼夜节律基因能够产生一个自我维持的自主振荡，其周期约为24小时。生物钟的一个主要特征是温度补偿。然而，温度补偿背后的机制仍然难以捉摸。先前的研究表明，单个生物钟基因可能决定几种模式生物中的温度补偿。为了了解每个单独的生物钟基因对温度补偿的影响，使用强大的基因编辑工具CRISPR/Cas9分别敲除了23个著名的哺乳动物生物钟基因以及相关基因。首先，作为示例敲除了相关基因，以验证通过CRISPR删除基因是有效且精确的。在小鼠成纤维细胞NIH3T3细胞中成功编辑了靶向22个基因的细胞系，脱靶分析表明这些基因被正确敲除。通过测量荧光素酶报告基因，在32.5℃和37℃这两种不同温度下记录了每个细胞系的昼夜节律周期。随后计算了每个细胞系的温度补偿系数Q。对这些细胞系的Q值估计表明，没有单个细胞系会对温度补偿产生不利影响。在较低温度下周期较长的细胞在较高温度下往往周期较短，而在较低温度下周期较短的细胞在较高温度下往往周期较长。因此，温度补偿是维持细胞内稳态的一项基本特性。我们进一步得出结论，温度补偿是一个复杂的基因调控系统，而不是由任何单个基因调控。我们还估计了这些细胞系的增殖率。在系统比较增殖率和昼夜节律周期后，我们发现细胞生长速率不依赖于昼夜节律周期。
