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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.

SUBMITTER: Mort RL

PROVIDER: S-EPMC4613862 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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

Mort Richard Lester RL Ford Matthew Jonathan MJ Sakaue-Sawano Asako A Lindstrom Nils Olof NO Casadio Angela A Douglas Adam Thomas AT Keighren Margaret Anne MA Hohenstein Peter P Miyawaki Atsushi A Jackson Ian James IJ

Cell cycle (Georgetown, Tex.) 20140101 17

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 ...[more]

PMID: 25486356

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Project description:Current genetic tools designed to target macrophages in vivo often target cells from all myeloid lineages. Therefore, we sought to generate a novel transgenic mouse which has a tamoxifen inducible Cre-recombinase under the control of the human CD68 promoter (hCD68-CreERT2). To test the efficiency and specificity of the of Cre-recombinase activity we crossed the hCD68-CreERT2 mice with a loxP-flanked STOP cassette red fluorescent protein variant (tdTomato) mouse. We established that orally dosing mice with 2 mg of tamoxifen for 5 consecutive days followed by a 5-day induction period resulted in robust expression of tdTomato in CD11b+ F4/80+ tissue resident macrophages. Using this induction protocol, we demonstrated tdTomato expression within peritoneal, liver and spleen macrophages and blood Ly6Clow monocytes. Importantly there was limited or no inducible tdTomato expression within other myeloid cells (neutrophils, monocytes, dendritic cells and eosinophils), T cells (CD4+ and CD8+) and B cells (CD19+). We also demonstrated that the level of tdTomato expression can be used as a marker to identify different populations of peritoneal and liver macrophages. We next assessed the longevity of tdTomato expression in peritoneal macrophages, liver and splenic macrophages and demonstrated high levels of tdTomato expression as long as 6 weeks after the last tamoxifen dose. Importantly, hCD68-CreERT2 expression is more restricted than that of LysM-Cre which has significant expression in major myeloid cell types (monocytes and neutrophils). To demonstrate the utility of this novel macrophage-specific Cre driver line we demonstrated tdTomato expression in recruited CD11b+CD64+F4/80+ monocyte-derived macrophages within the atherosclerotic lesions of AAV8-mPCSK9 treated mice, with limited expression in recruited neutrophils. In developing this new hCD68-CreERT2 mouse we have a tool that allows us to target tissue resident macrophages, with the advantage of not targeting other myeloid cells namely neutrophils and inflammatory monocytes.

Project description:In the mouse retina, horizontal cells form an electrically coupled network and provide feedback signals to photoreceptors and feedforward signals to bipolar cells. Thereby, horizontal cells contribute to gain control at the first visual synapse and to the antagonistic organization of bipolar and ganglion cell receptive fields. However, the nature of horizontal cell output remains a matter of debate, just as the exact contribution of horizontal cells to center-surround antagonism. To facilitate studying horizontal cell function, we developed a knockin mouse line which allows ablating genes exclusively in horizontal cells. This knockin line expresses a Cre recombinase under the promoter of connexin57 (Cx57), a gap junction protein only expressed in horizontal cells. Consistently, in Cx57+/Cre mice, Cre recombinase is expressed in almost all horizontal cells (>99%) and no other retinal neurons. To test Cre activity, we crossbred Cx57+/Cre mice with a mouse line in which exon 11 of the coding sequence for the ionotropic glutamate receptor subunit GluA4 was flanked by two loxP sites (GluA4fl/fl). In GluA4fl/fl:Cx57+/Cre mice, GluA4 immunoreactivity was significantly reduced (∼ 50%) in the outer retina where horizontal cells receive photoreceptor inputs, confirming the functionality of the Cre/loxP system. Whole-cell patch-clamp recordings from isolated horizontal cell somata showed a reduction of glutamate-induced inward currents by ∼ 75%, suggesting that the GluA4 subunit plays a major role in mediating photoreceptor inputs. The persistent current in GluA4-deficient cells is mostly driven by AMPA and to a very small extent by kainate receptors as revealed by application of the AMPA receptor antagonist GYKI52466 and concanavalin A, a potentiator of kainate receptor-mediated currents. In summary, the Cx57+/Cre mouse line provides a versatile tool for studying horizontal cell function. GluA4fl/fl:Cx57+/Cre mice, in which horizontal cells receive less excitatory input, can thus be used to analyze the contribution of horizontal cells to retinal processing.

Dataset Information

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

Publications

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

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