An In Vitro ES Cell-Based Clock Recapitulation Assay Model Identifies CK2a as an Endogenous Clock Regulator

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 (CKId) induced,3 hours longer period-length of circadian rhythm than the wild type, which was compatible with recently reported results using CKId null mice. In addition, this assay system also revealed that a Casein Kinase 2 alpha subunit (CK2a) 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 CK2a gene. Since CK2a deficient mice are embryonic lethal, this in vitro assay system represents the genetic evidence showing an essential role of CK2a in the mammalian circadian clock. This assay was successfully applied for the phenotype analysis of homozygou