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Cold Spring Harb Protoc. 2014 May 1;2014(5). pii: pdb.prot080408. doi: 10.1101/pdb.prot080408.

Visualizing spatiotemporal dynamics of multicellular cell-cycle progressions with fucci technology.

Abstract

The visualization of cell-cycle behavior of individual cells within complex tissues presents an irresistible challenge to biologists studying multicellular structures. However, the transition from G1 to S in the cell cycle is difficult to monitor despite the fact that the process involves the critical decision to initiate a new round of DNA replication. Here, we use ubiquitination oscillators that control cell-cycle transitions to develop genetically encoded fluorescent probes for cell-cycle progression. Fucci (fluorescent ubiquitination-based cell-cycle indicator) probes exploit the regulation of cell-cycle-dependent ubiquitination to effectively label individual nuclei in G1 phase red, and those in S/G2/M phases green. Cultured cells and transgenic mice constitutively expressing the probes have been generated, such that every cell nucleus shows either red or green fluorescence. This protocol details two experiments that use biological samples expressing Fucci probes. One experiment involves time-lapse imaging of cells stably expressing a Fucci derivative (Fucci2), which allows for the exploration of the spatiotemporal patterns of cell-cycle dynamics during structural and behavioral changes of cultured cells. The other experiment involves large-field, high-resolution imaging of fixed sections of Fucci transgenic mouse embryos, which provides maps that illustrate cell proliferation versus differentiation in various developing organs.

PMID:
24786503
DOI:
10.1101/pdb.prot080408
[Indexed for MEDLINE]

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