title: An in vitro ES cell-based clock recapitulation assay model identifies CK2α as an endogenous clock regulator.
abstract: We previously reported emergence and disappearance of circadian molecular oscillations during differentiation of mouse embryonic stem (ES) cells and reprogramming of differentiated cells, respectively. Here we present a robust and stringent in vitro circadian clock formation assay that recapitulates in vivo circadian phenotypes. This assay system first confirmed that a mutant ES cell line lacking Casein Kinase I delta (CKIδ) induced ∼3 hours longer period-length of circadian rhythm than the wild type, which was compatible with recently reported results using CKIδ null mice. In addition, this assay system also revealed that a Casein Kinase 2 alpha subunit (CK2α) homozygous mutant ES cell line developed significantly longer (about 2.5 hours) periods of circadian clock oscillations after in vitro or in vivo differentiation. Moreover, revertant ES cell lines in which mutagenic vector sequences were deleted showed nearly wild type periods after differentiation, indicating that the abnormal circadian period of the mutant ES cell line originated from the mutation in the CK2α gene. Since CK2α deficient mice are embryonic lethal, this in vitro assay system represents the genetic evidence showing an essential role of CK2α in the mammalian circadian clock. This assay was successfully applied for the phenotype analysis of homozygous mutant ES cells, demonstrating that an ES cell-based in vitro assay is available for circadian genetic screening.
authors.affiliation: Department of Physiology and Systems Bioscience, Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Umemura
authors.forename: Junko
authors.initials: J
authors.lastname: Yoshida
authors.forename: Masashi
authors.initials: M
authors.lastname: Wada
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Tsuchiya
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Minami
authors.forename: Hitomi
authors.initials: H
authors.lastname: Watanabe
authors.forename: Gen
authors.initials: G
authors.lastname: Kondoh
authors.forename: Junji
authors.initials: J
authors.lastname: Takeda
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Inokawa
authors.forename: Kyoji
authors.initials: K
authors.lastname: Horie
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Yagita
mesh_terms: D000818:Animals; D047390:Casein Kinase II / Q000502:physiology*; D048148:Casein Kinase Idelta / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D002678:Chimera / Q000502:physiology; D057906:Circadian Clocks; D002940:Circadian Rhythm; D018920:Coculture Techniques; D053595:Embryonic Stem Cells / Q000201:enzymology* / Q000502:physiology; D061252:Feeder Cells; D015870:Gene Expression; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008822:Mice, Transgenic; D016254:Mutagenesis, Insertional
chemical_list: D047390:Casein Kinase II; D048148:Casein Kinase Idelta