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 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.

White paper on guidelines concerning enteric nervous system stem cell therapy for enteric neuropathies

“…Finally, to unravel the above mentioned influence of different culture conditions and sorting strategies on in vitro stem cell behavior it might be helpful to analyze enteric neural crest-derived cells using recently developed transgenic reporter mouse models to track cell cycle (Abe et al, 2013; Mort et al, 2014), stress responses (Thorp et al, 2011), chromosomal instability (Balbach and Boiani, 2015) or stem cell signaling (Ferrer-Vaquer et al, 2010) (Balaskas et al, 2012) in vitro and in vivo . Furthermore, embryonic development can be simulated in vitro using recent (human) pluripotent stem cell technologies to understand crucial molecular checkpoints for differentiation towards enteric neural progenitors and fully mature neural cells (Kawaguchi et al, 2010; Sasselli et al, 2012a).…”

Blue intensity matters for cell cycle profiling in fluorescence DAPI-stained images

“…29 The possibility to use exogenously expressed cell cycle markers, as Fucci-assisted reporter systems (reviewed in 30) facilitated the quantitative analysis of cell cycle status, especially when studying complex systems as whole animals. [31][32][33] Despite the fact that the development of these fluorescence cell cycle sensors came to revolutionize our ability to see the cell cycle, especially in live-cell imaging-based techniques, these genetically engineered systems (i) do not tackle the bleed-through problems inherent to conventional fluorochrome-dependent analyses, (ii) are time-consuming to establish and (iii) display some pitfalls in the identification of some cell cycle phases. 30 Conversely, the image-based framework presented here depends only on cell nucleus staining to profile the cell cycle of unsynchronized 2D cell cultures.…”

Tissue-Specific Cell Cycle Indicator Reveals Unexpected Findings for Cardiac Myocyte Proliferation

“…10 Initial iterations of this kind of reporter were generated using a cytomegalo-virus-based enhancer, were subject to issues with transgene expression, and were not cell type–specific, although in more recent iterations, tissue conditional expression is possible. 11,12 Investigation of cardiac myocyte proliferation with these reporters was recently reported. 13 Here, we undertook a distinct approach and generated a CyclinA2-reporter fusion protein under the control of the endogenous CyclinA2 locus.…”