RRC ID 31998
Author Yo M, Sakaue-Sawano A, Noda S, Miyawaki A, Miyoshi H.
Title Fucci-guided purification of hematopoietic stem cells with high repopulating activity.
Journal Biochem. Biophys. Res. Commun.
Abstract 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(-/low)c-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.
Volume 457(1)
Pages 7-11
Published 2015-1-30
DOI 10.1016/j.bbrc.2014.12.074
PII S0006-291X(14)02256-6
PMID 25534850
MeSH Animals Cell Cycle* Cell Separation / methods* Flow Cytometry Fluorescence Fluorescent Dyes / metabolism* Hematopoietic Stem Cells / cytology* Hematopoietic Stem Cells / metabolism Mice, Transgenic Transgenes Ubiquitination*
IF 2.559
Times Cited 4
Mice C57BL/6-Ly5.1(RBRC00144) B6.Cg-Tg(FucciS/G2/M)#474Bsi Tg(FucciG1)#610Bsi(RBRC02817) B6.Cg-Tg(FucciS/G2/M)#474Bsi(RBRC02704) B6.Cg-Tg(FucciG1)#610Bsi(RBRC02708)