Final published version
Licence: CC BY-NC
Research output: Contribution to Journal/Magazine › Journal article › peer-review
Research output: Contribution to Journal/Magazine › Journal article › peer-review
}
TY - JOUR
T1 - Fucci2a
T2 - a bicistronic cell cycle reporter that allows Cre mediated tissue specific expression in mice
AU - Mort, Richard Lester
AU - Ford, Matthew Jonathan
AU - Sakaue-Sawano, Asako
AU - Lindstrom, Nils Olof
AU - Casadio, Angela
AU - Douglas, Adam Thomas
AU - Keighren, Margaret Anne
AU - Hohenstein, Peter
AU - Miyawaki, Atsushi
AU - Jackson, Ian James
PY - 2014
Y1 - 2014
N2 - 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.
AB - 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.
KW - 3T3 Cells
KW - Animals
KW - Cell Cycle
KW - Cell Proliferation
KW - Cell Survival
KW - Embryo, Mammalian
KW - Embryonic Stem Cells
KW - G1 Phase
KW - Gene Expression
KW - Genes, Reporter
KW - Humans
KW - Integrases
KW - Kidney
KW - Luminescent Proteins
KW - Lung
KW - Mice
KW - Mitosis
KW - Morphogenesis
KW - Organ Specificity
KW - Time Factors
KW - Time-Lapse Imaging
KW - Journal Article
KW - Research Support, Non-U.S. Gov't
U2 - 10.4161/15384101.2015.945381
DO - 10.4161/15384101.2015.945381
M3 - Journal article
C2 - 25486356
VL - 13
SP - 2681
EP - 2696
JO - Cell Cycle (Georgetown, Tex.)
JF - Cell Cycle (Georgetown, Tex.)
SN - 1538-4101
IS - 17
ER -