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Cell Cycle. 2014;13(17):2681-96. doi: 10.4161/15384101.2015.945381.

Fucci2a: a bicistronic cell cycle reporter that allows Cre mediated tissue specific expression in mice.

Author information

1
a MRC Human Genetics Unit; MRC IGMM ; University of Edinburgh; Western General Hospital Edinburgh ; Scotland , UK.

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.

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

PMID:
25486356
PMCID:
PMC4613862
DOI:
10.4161/15384101.2015.945381
[Indexed for MEDLINE]
Free PMC Article

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