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

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    Rights statement: © 2014 The Author(s). Published with license by Taylor & Francis Group, LLC© Richard Lester Mort, Matthew Jonathan Ford, Asako Sakaue-Sawano, Nils Olof Lindstrom, Angela Casadio, Adam Thomas Douglas, Margaret Anne Keighren, Peter Hohenstein, Atsushi Miyawaki, and Ian James Jackson This is an Open Access article distributed under the terms of the Creative Commons Attribution-Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.

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Original languageEnglish
Pages (from-to)2681-2696
Number of pages16
JournalCell Cycle
Volume13
Issue number17
DOIs
StatePublished - 1 Sep 2014

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 reengineered 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.

Research areas

  • Cell cycle, Fucci, Fucci2, Fucci2a, Kidney, Lung, Melanoblast

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