title: Fucci2a: a bicistronic cell cycle reporter that allows Cre mediated tissue specific expression in mice.
abstract: Markers of cell cycle stage allow estimation of cell cycle dynamics in cell culture and during embryonic development. The Fucci system incorporates genetically encoded probes that highlight G1 and S/G2/M phases of the cell cycle allowing live imaging. However the available mouse models that incorporate Fucci are beset by problems with transgene inactivation, varying expression level, lack of conditional potential and/or the need to maintain separate transgenes-there is no transgenic mouse model that solves all these problems. To address these shortfalls we re-engineered the Fucci system to create 2 bicistronic Fucci variants incorporating both probes fused using the Thosea asigna virus 2A (T2A) self cleaving peptide. We characterize these variants in stable 3T3 cell lines. One of the variants (termed Fucci2a) faithfully recapitulated the nuclear localization and cell cycle stage specific florescence of the original Fucci system. We go on to develop a conditional mouse allele (R26Fucci2aR) carefully designed for high, inducible, ubiquitous expression allowing investigation of cell cycle status in single cell lineages within the developing embryo. We demonstrate the utility of R26Fucci2aR for live imaging by using high resolution confocal microscopy of ex vivo lung, kidney and neural crest development. Using our 3T3 system we describe and validate a method to estimate cell cycle times from relatively short time-lapse sequences that we then apply to our neural crest data. The Fucci2a system and the R26Fucci2aR mouse model are compelling new tools for the investigation of cell cycle dynamics in cell culture and during mouse embryonic development.
authors.affiliation: a MRC Human Genetics Unit; MRC IGMM ; University of Edinburgh; Western General Hospital Edinburgh ; Scotland , UK.
authors.forename: Richard Lester
authors.initials: RL
authors.lastname: Mort
authors.forename: Matthew Jonathan
authors.initials: MJ
authors.lastname: Ford
authors.forename: Asako
authors.initials: A
authors.lastname: Sakaue-Sawano
authors.forename: Nils Olof
authors.initials: NO
authors.lastname: Lindstrom
authors.forename: Angela
authors.initials: A
authors.lastname: Casadio
authors.forename: Adam Thomas
authors.initials: AT
authors.lastname: Douglas
authors.forename: Margaret Anne
authors.initials: MA
authors.lastname: Keighren
authors.forename: Peter
authors.initials: P
authors.lastname: Hohenstein
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyawaki
authors.forename: Ian James
authors.initials: IJ
authors.lastname: Jackson
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002453:Cell Cycle; D049109:Cell Proliferation; D002470:Cell Survival; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D016193:G1 Phase; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D019426:Integrases / Q000378:metabolism*; D007668:Kidney / Q000196:embryology; D008164:Luminescent Proteins / Q000378:metabolism; D008168:Lung / Q000196:embryology; D051379:Mice; D008938:Mitosis; D009024:Morphogenesis; D009928:Organ Specificity; D013997:Time Factors; D059008:Time-Lapse Imaging; D000097573:Red Fluorescent Protein
chemical_list: D019426:Integrases; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein; C045073:Cre recombinase
keywords: BrdU, 5-bromo-2′-deoxyuridine; DAPI, 4′, 6-diamidino-2-phenylindole; DMEM, Dulbeccos modified eagle medium; ECACC, European Collection of Cell Cultures; EMMA, European Mouse Mutant Archive; FACS, Fluorescence-activated cell sorting; Fucci; Fucci, Fluorescent Ubiquitination-based Cell Cycle Indicator; Fucci2; Fucci2a; GMEM, Glasgow minimum essential medium; IRES, Internal ribosomal entry site; LIF, leukemia inhibitory factor; RBDB, Riken Bioresource Center DNA Bank; T2A, Thosea asigna virus 2A peptide; cell cycle; hESC, Human embryonic stem cell; kidney; lung; mAG, Monomeric Azami Green; mESC, Mouse embryonic stem cell; mKO2, Monomeric Kusabira Orange; melanoblast