Visualization of cell cycle in mouse embryos with Fucci2 reporter directed by Rosa26 promoter

Abstract

SUMMARYFucci technology makes possible the distinction between live cells in the G 1 and S/G 2 /M phases by dual-color imaging. This technology relies upon ubiquitylation-mediated proteolysis, and transgenic mice expressing Fucci provide a powerful model system with which to study the coordination of the cell cycle and development. The mice were initially generated using the CAG promoter; lines expressing the G 1 and S/G 2 /M phase probes that emitted orange (mKO2) and green (mAG) fluorescence, respectively, were separately constructed. Owing to cell type-biased strength of the CAG promoter as well as the positional effects of random transgenesis, however, we noticed some variability in Fucci expression levels. To control more reliably the expression of cell cycle probes, we used different genetic approaches to create two types of reporter mouse lines with Fucci2 and Rosa26 transcriptional machinery. Fucci2 is a recently developed Fucci derivative, which emits red (mCherry) and green (mVenus) fluorescence and provides better color contrast than Fucci. A new transgenic line, R26p-Fucci2, utilizes the Rosa26 promoter and harbors the G 1 and S/G 2 /M phase probes in a single transgene to preserve their co-inheritance. In the other R26R-Fucci2 approach, the two probes are incorporated into Rosa26 locus conditionally. The Cre-mediated loxP recombination technique thus allows researchers to design cell-type-specific Fucci2 expression. By performing time-lapse imaging experiments using R26p-Fucci2 and R26-Fucci2 in which R26R-Fucci2 had undergone germline loxP recombination, we demonstrated the great promise of these mouse reporters for studying cell cycle behavior in vivo.

FUCCI tracking shows cell‐cycle‐dependent Neurog3 variation in pancreatic progenitors

“…Unexpectedly, we detected significant residual cytoplasmic mVenus-hGem (1/110) fluorescence in post-mitotic, actively delaminating, Muc1 + endocrine-committed Neurog3 TA.HI cells that showed the expected high mKO2-hCdt1 (30/120) signal (Figure 3A). This observation was different from previous reports on the FUCCI reporter, where mVenus-hGem (1/110) was mostly degraded soon after M-phase, becoming absent by the time of mKO2-hCdt (30/120) detection in early G 1 (Abe et al, 2013; Sakaue-Sawano et al, 2008). This unexpected overlap between mVenus-hGem (1/110) (cytoplasmic) and mKO2-hCdt1 (30/120) (nuclear) signals was essentially completely restricted to the actively delaminating post-mitotic Sox9 − Neurog3 TA.HI population, and >85% of the cells within this pool displayed this co-positivity.…”

Proliferative heterogeneity of murine epithelial cells in the adult mammary gland

“…We employed the Fucci2 reporter mouse to visualize mammary epithelial cells in specific phases of the cell cycle. In the Fucci2 system, mCherry-hCdt1 (30/120) is expressed during G1, while mVenus-hGem (1/110) is expressed during S/G2/M phase of the cell cycle 7 . Additionally, both colours disappear during cytokinesis and very early in G1 7 .…”

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

“…Fucci2a performs identically to the original Fucci and Fucci2 probes, 11,14 but offers the key advantage of being composed of a single genetic construct. Table 1 highlights the resources developed during this study and the repositories in which they have been housed.…”