title: Isolation of teleost primordial germ cells using flow cytometry.
abstract: Primordial germ cells (PGCs) generate gametes, the only cells that can transmit genetic information to the next generation. A previous report demonstrated that a fusion construct of green fluorescent protein (gfp) and zebrafish nos 1 3UTR mRNA could be used to label PGCs in a number of fish species. Here, we sought to exploit this labeling strategy to isolate teleost PGCs by flow cytometry (FCM), and to use these isolated PGCs to examine germ cell migration to the gonadal region. In zebrafish, medaka and goldfish, the PGCs were labeled by injecting the gfp-nos1 3UTR mRNA into 1- 4 cell embryos. When the embryos had developed to the somitogenesis or later stages, they were enzymatically disaggregated and GFP positive cells isolated using FCM. PGCs in the different species clustered in the same segments of the FCM scatter diagrams for total embryonic cells produced by plotting the forward scatter intensity against GFP intensity. In situ hybridization showed that the sorted zebrafish cells expressed vasa RNA in their cytoplasm, suggesting that they were PGCs. When the migration ability of the sorted cells from zebrafish was examined in an in vivo transplantation experiment, approximately 30% moved to the gonadal region of host embryos. These observations demonstrate that PGCs can be isolated without use of transgenic fishes and that the isolated PGCs retain the ability to migrate. Our data indicate that this technique will be of value for isolating PGCs from a range of fish species.
authors.affiliation: Nanae Fresh Water laboratory, Field Science Center for Northern Biosphere, Hokkaido University, Hakodate, Hokkaido, Japan. goto-kazeto@hokudai-nanae.jp
authors.forename: Rie
authors.initials: R
authors.lastname: Goto-Kazeto
authors.forename: Taiju
authors.initials: T
authors.lastname: Saito
authors.forename: Misae
authors.initials: M
authors.lastname: Takagi
authors.forename: Katsutoshi
authors.initials: K
authors.lastname: Arai
authors.forename: Etsuro
authors.initials: E
authors.lastname: Yamaha
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D002465:Cell Movement; D002469:Cell Separation / Q000379:methods*; D053487:DEAD-box RNA Helicases / Q000235:genetics; D005434:Flow Cytometry; D015870:Gene Expression; D005854:Germ Cells / Q000166:cytology* / Q000637:transplantation; D006054:Goldfish / Q000196:embryology*; D006066:Gonads / Q000196:embryology; D049452:Green Fluorescent Proteins; D017403:In Situ Hybridization; D009990:Oryzias / Q000196:embryology*; D012333:RNA, Messenger / Q000235:genetics; D016601:RNA-Binding Proteins; D013194:Staining and Labeling; D015027:Zebrafish / Q000196:embryology*; D029961:Zebrafish Proteins / Q000235:genetics
chemical_list: D020413:3' Untranslated Regions; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D029961:Zebrafish Proteins; C440015:nanos3 protein, zebrafish; D049452:Green Fluorescent Proteins; C498289:ddx4 protein, zebrafish; D053487:DEAD-box RNA Helicases