Knockout-Rescue Embryonic Stem Cell-Derived Mouse Reveals Circadian-Period Control by Quality and Quantity of CRY1
Knockout-Rescue Embryonic Stem Cell-Derived Mouse Reveals Circadian-Period Control by Quality and Quantity of CRY1
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DOI：
10.1016/j.molcel.2016.11.022
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发表时间：
2017-01-05
期刊：
影响因子：
16
通讯作者：
Ueda, Hiroki R.
中科院分区：
文献类型：
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作者：
Ode, Koji L.;Ukai, Hideki;Ueda, Hiroki R.
为了对生物体中的分子特性进行全面的表征，我们建立了一种有效的方法，在一代内培育出基因敲除(KO)救援小鼠。我们应用这种方法在Cry1(-/-)：Cry2(-/-)背景下获得了20株几乎完全由胚胎干细胞(ESC)来源的小鼠(“ES小鼠”)，它们是在Cry1(-/-)：Cry2(-/-)背景下被拯救的野生型和突变型Cry1基因。一系列模拟磷酸化和非模拟磷酸化的CRY1突变体表明，CRY1的多位点磷酸化可以作为哺乳动物生物钟的累积计时器。KO-RESAVE ES小鼠还发现CRY1-PER2相互作用使小鼠具有强大的昼夜节律性。令人惊讶的是，与经典转录/翻译反馈环的理论预测相反，CRY1柔性P环和C-LID结构域周围的残基决定了昼夜节律，而不改变CRY1的降解速率。这些结果表明，CRY1通过降解依赖和非独立的途径来决定昼夜节律。
To conduct comprehensive characterization of molecular properties in organisms, we established an efficient method to produce knockout (KO)rescue mice within a single generation. We applied this method to produce 20 strains of almost completely embryonic stem cell (ESC)-derived mice ("ES mice'') rescued with wild-type and mutant Cry1 gene under a Cry1(-/-):Cry2(-/-) background. A series of both phosphorylation-mimetic and non-phosphorylation-mimetic CRY1 mutants revealed that multisite phosphorylation of CRY1 can serve as a cumulative timer in the mammalian circadian clock. KO-rescue ES mice also revealed that CRY1-PER2 interaction confers a robust circadian rhythmicity in mice. Surprisingly, in contrast to theoretical predictions from canonical transcription/translation feedback loops, the residues surrounding the flexible P loop and C-lid domains of CRY1 determine circadian period without changing the degradation rate of CRY1. These results suggest that CRY1 determines circadian period through both its degradation-dependent and -independent pathways.