Fluorescence activated cell sorting of live female germ cells and somatic cells of the mouse fetal gonad based on forward and side scattering.
Analysis of female mammalian germ cells has been hindered by difficulties in isolating high purity germ cell populations from embryonic and fetal gonads. Meiotic prophase stage oocytes are particularly difficult to isolate due to the lack of suitable surface markers. Oct4 promoter driven GFP expression has been used to distinguish germ cells/oocytes (GFP positive) from somatic cells (GFP negative), however, the requirement for transgenic animals has limited the use of this technique. We analyzed the side- and forward scattering properties of living cell populations obtained from fetal ovaries of Oct4-GFP transgenic and wild-type mice. On the basis of these measurements, we defined criteria that allow the discrimination and identification of germ cells and somatic cells within cell suspensions of nontransgenic female fetal gonads. The described method is suitable for the isolation of populations of germ cells and somatic cells of higher than 90% purity. We also demonstrated that the sorted cells can be used in downstream immunofluorescence and RT-PCR applications. Hence, we conclude that side and forward scattering based sorting of female germ cells is a valuable tool that will benefit the understanding of female gametogenesis.
Wojtasz
Lukasz
L
Institute of Physiological Chemistry, Dresden University of Technology, Fiedlerstr. 42, 01307 Dresden, Germany.
Daniel
Katrin
K
Toth
Attila
A
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D005854:Germ Cells / Q000166:cytology* / Q000378:metabolism; D006066:Gonads / Q000166:cytology / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D010053:Ovary / Q000166:cytology* / Q000378:metabolism
D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D049452:Green Fluorescent Proteins