title: Visualization of cell cycle in mouse embryos with Fucci2 reporter directed by Rosa26 promoter.
abstract: Fucci technology makes possible the distinction between live cells in the G(1) and S/G(2)/M phases by dual-color imaging. This technology relies upon ubiquitylation-mediated proteolysis, and transgenic mice expressing Fucci provide a powerful model system with which to study the coordination of the cell cycle and development. The mice were initially generated using the CAG promoter; lines expressing the G(1) and S/G(2)/M phase probes that emitted orange (mKO2) and green (mAG) fluorescence, respectively, were separately constructed. Owing to cell type-biased strength of the CAG promoter as well as the positional effects of random transgenesis, however, we noticed some variability in Fucci expression levels. To control more reliably the expression of cell cycle probes, we used different genetic approaches to create two types of reporter mouse lines with Fucci2 and Rosa26 transcriptional machinery. Fucci2 is a recently developed Fucci derivative, which emits red (mCherry) and green (mVenus) fluorescence and provides better color contrast than Fucci. A new transgenic line, R26p-Fucci2, utilizes the Rosa26 promoter and harbors the G(1) and S/G(2)/M phase probes in a single transgene to preserve their co-inheritance. In the other R26R-Fucci2 approach, the two probes are incorporated into Rosa26 locus conditionally. The Cre-mediated loxP recombination technique thus allows researchers to design cell-type-specific Fucci2 expression. By performing time-lapse imaging experiments using R26p-Fucci2 and R26-Fucci2 in which R26R-Fucci2 had undergone germline loxP recombination, we demonstrated the great promise of these mouse reporters for studying cell cycle behavior in vivo.
authors.affiliation: Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Developmental Biology (CDB), Kobe 650-0047, Japan.
authors.forename: Takaya
authors.initials: T
authors.lastname: Abe
authors.forename: Asako
authors.initials: A
authors.lastname: Sakaue-Sawano
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kiyonari
authors.forename: Go
authors.initials: G
authors.lastname: Shioi
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Inoue
authors.forename: Toshitaka
authors.initials: T
authors.lastname: Horiuchi
authors.forename: Kazuki
authors.initials: K
authors.lastname: Nakao
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyawaki
authors.forename: Shinichi
authors.initials: S
authors.lastname: Aizawa
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Fujimori
mesh_terms: D000818:Animals; D002453:Cell Cycle / Q000235:genetics*; D004622:Embryo, Mammalian / Q000166:cytology* / Q000502:physiology; D005453:Fluorescence; D016193:G1 Phase / Q000235:genetics; D016195:G2 Phase / Q000235:genetics; D017930:Genes, Reporter; D051379:Mice; D008822:Mice, Transgenic; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011506:Proteins / Q000235:genetics*; D022661:RNA, Untranslated; D016196:S Phase / Q000235:genetics; D059008:Time-Lapse Imaging / Q000379:methods*; D054875:Ubiquitination / Q000235:genetics
chemical_list: C110853:Gt(ROSA)26Sor non-coding RNA, mouse; D011506:Proteins; D022661:RNA, Untranslated