Purification and analysis of in vivo-differentiated oligodendrocytes expressing the green fluorescent protein.
A complete understanding of the molecular mechanisms involved in the formation and repair of the central nervous system myelin sheath requires an unambiguous identification and isolation of in vivo-differentiated myelin-forming cells. In order to develop a novel tool for the analysis of in vivo-differentiated oligodendrocytes, we generated transgenic mice expressing a red-shifted variant of the green fluorescent protein under the control of the proteolipid protein promoter. We demonstrate here that green fluorescent protein-derived fluorescence in the central nervous system of 9-day- to 7-week-old mice is restricted to mature oligodendrocytes, as determined by its spatiotemporal appearance and by both immunocytochemical and electrophysiological criteria. Green fluorescent protein-positive oligodendrocytes could easily be visualized in live and fixed tissue. Furthermore, we show that this convenient and reliable identification now allows detailed physiological analyses of differentiated oligodendrocytes in situ. In addition, we developed a novel tissue culture system for in vivo-differentiated oligodendrocytes. Initial data using this system indicate that, for oligodendrocytes isolated after differentiation in vivo, as yet unidentified factors secreted by astrocytes are necessary for survival and/or reappearance of a mature phenotype in culture.
Fuss
B
B
Department of Neurosciences, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio, USA.
Mallon
B
B
Phan
T
T
Ohlemeyer
C
C
Kirchhoff
F
F
Nishiyama
A
A
Macklin
W B
WB
D000818:Animals; D001483:Base Sequence; D002469:Cell Separation; D017931:DNA Primers; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008164:Luminescent Proteins / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D018991:Myelin Proteolipid Protein / Q000235:genetics; D009836:Oligodendroglia / Q000166:cytology / Q000378:metabolism*; D010641:Phenotype
D017931:DNA Primers; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D018991:Myelin Proteolipid Protein; D049452:Green Fluorescent Proteins