TY - JOUR
T1 - Effect of Multiple Clock Gene Ablations on the Circadian Period Length and Temperature Compensation in Mammalian Cells
AU - Tsuchiya, Yoshiki
AU - Umemura, Yasuhiro
AU - Minami, Yoichi
AU - Koike, Nobuya
AU - Hosokawa, Toshihiro
AU - Hara, Masayuki
AU - Ito, Hiroshi
AU - Inokawa, Hitoshi
AU - Yagita, Kazuhiro
N1 - Funding Information:
We thank Drs. Munehiro Ohashi and Tasuku Hara (Kyoto Prefectural University of Medicine) for their valuable discussions and technical support. The authors were supported in part by a Japan Science and Technology Agency Precursory Research for Embryonic Science and Technology program (to K.Y.), grants-in-aid for scientific research from the Japan Society for the Promotion of Science (to K.Y., Y.T., and Y.U.), and the Takeda Science Foundation (to K.Y.).
Publisher Copyright:
© 2015 The Author(s).
PY - 2016/2/1
Y1 - 2016/2/1
N2 - Most organisms have cell-autonomous circadian clocks to coordinate their activity and physiology according to 24-h environmental changes. Despite recent progress in circadian studies, it is not fully understood how the period length and the robustness of mammalian circadian rhythms are determined. In this study, we established a series of mouse embryonic stem cell (ESC) lines with single or multiplex clock gene ablations using the CRISPR/Cas9-based genome editing method. ESC-based in vitro circadian clock formation assay shows that the CRISPR-mediated clock gene disruption not only reproduces the intrinsic circadian molecular rhythms of previously reported mice tissues and cells lacking clock genes but also reveals that complexed mutations, such as CKIm/m:CKI+/m:Cry2m/m mutants, exhibit an additively lengthened circadian period. By using these mutant cells, we also investigated the relation between period length alteration and temperature compensation. Although CKI-deficient cells slightly affected the temperature insensitivity of period length, we demonstrated that the temperature compensation property is largely maintained in all mutants. These results show that the ESC-based assay system could offer a more systematic and comprehensive approach to the genotype-chronotype analysis of the intracellular circadian clockwork in mammals.
AB - Most organisms have cell-autonomous circadian clocks to coordinate their activity and physiology according to 24-h environmental changes. Despite recent progress in circadian studies, it is not fully understood how the period length and the robustness of mammalian circadian rhythms are determined. In this study, we established a series of mouse embryonic stem cell (ESC) lines with single or multiplex clock gene ablations using the CRISPR/Cas9-based genome editing method. ESC-based in vitro circadian clock formation assay shows that the CRISPR-mediated clock gene disruption not only reproduces the intrinsic circadian molecular rhythms of previously reported mice tissues and cells lacking clock genes but also reveals that complexed mutations, such as CKIm/m:CKI+/m:Cry2m/m mutants, exhibit an additively lengthened circadian period. By using these mutant cells, we also investigated the relation between period length alteration and temperature compensation. Although CKI-deficient cells slightly affected the temperature insensitivity of period length, we demonstrated that the temperature compensation property is largely maintained in all mutants. These results show that the ESC-based assay system could offer a more systematic and comprehensive approach to the genotype-chronotype analysis of the intracellular circadian clockwork in mammals.
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U2 - 10.1177/0748730415613888
DO - 10.1177/0748730415613888
M3 - Article
C2 - 26511603
AN - SCOPUS:84954151551
SN - 0748-7304
VL - 31
SP - 48
EP - 56
JO - Journal of Biological Rhythms
JF - Journal of Biological Rhythms
IS - 1
ER -