| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 1928248 | Biochemical and Biophysical Research Communications | 2015 | 5 Pages |
•We generated transgenic mice expressing fluorescent ubiquitination-based cell cycle indicator (Fucci) probes.•Transgenic mice expressed Fucci probes at high levels in most hematopoietic cell populations.•The fluorescence intensity of Fucci can be used for purification of hematopoietic stem cells.
Fluorescent ubiquitination-based cell cycle indicator (Fucci) technology utilizing the cell cycle-dependent proteolysis of ubiquitin oscillators enables visualization of cell cycle progression in living cells. The Fucci probe consists of two chimeric fluorescent proteins, FucciS/G2/M and FucciG1, which label the nuclei of cells in S/G2/M phase green and those in G1 phase red, respectively. In this study, we generated Fucci transgenic mice and analyzed transgene expression in hematopoietic cells using flow cytometry. The FucciS/G2/M-#474 and FucciG1-#639 mouse lines exhibited high-level transgene expression in most hematopoietic cell populations. The FucciG1-#610 line expressed the transgene at high levels predominantly in the hematopoietic stem cell (HSC) population. Analysis of the HSC (CD34−KSL: CD34−/lowc-Kit+Sca-1+lineage marker−) population in the transgenic mice expressing both FucciS/G2/M and FucciG1 (#474/#610) confirmed that more than 95% of the cells were in G0/G1 phase, although the FucciG1(red) intensity was heterogeneous. An in vivo competitive repopulation assay revealed that repopulating activity resided largely in the FucciG1(red)high fraction of CD34−KSL cells. Thus, the CD34−KSL HSC population can be further purified on the basis of the Fucci intensity.
