title: Mass isolation of primordial germ cells from transgenic rainbow trout carrying the green fluorescent protein gene driven by the vasa gene promoter.
abstract: Mass isolation of live primordial germ cells (PGCs) was demonstrated for the first time in ectothermal vertebrates. To establish a stem cell-mediated gene transfer system in fish, a stem cell line that retains the ability to develop into gametes is necessary. PGCs are well suited for use as the initial material for such a stem cell line. We established transgenic rainbow trout (Oncorhynchus mykiss) strains carrying the green fluorescent protein (GFP) gene driven by a rainbow trout vasa-like gene (RtVLG) promoter/enhancer. Because GFP expression was specific to the PGCs, PGCs were successfully visualized in all developmental stages examined. Isolated genital ridges containing GFP-labeled PGCs were enzymatically dissociated. To isolate PGCs from the complex pools of dissociated genital ridges, GFP-labeled cells were sorted by flow cytometry. The sorted GFP-positive cells were large and round with a large nucleus, typical characters of PGC morphology. The expression of RtVLG was detected only in the GFP-positive cell population, confirming that these cells were PGCs. This simple and efficient technique to purify a large number of viable PGCs opens the way for establishing a stem cell line, which can differentiate into the germline. The purified PGCs would also be a novel tool for cellular and molecular study of vertebrate germline stem cells.
authors.affiliation: Department of Aquatic Biosciences, Tokyo University of Fisheries, Minato-ku, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Takeuchi
authors.forename: Goro
authors.initials: G
authors.lastname: Yoshizaki
authors.forename: Terumasa
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Toshio
authors.initials: T
authors.lastname: Takeuchi
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods*; D005260:Female; D005434:Flow Cytometry; D015870:Gene Expression; D005854:Germ Cells / Q000737:chemistry / Q000166:cytology*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics*; D008297:Male; D017686:Oncorhynchus mykiss / Q000196:embryology / Q000235:genetics; D011401:Promoter Regions, Genetic; D020365:RNA Helicases / Q000235:genetics*; D013234:Stem Cells
chemical_list: D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D020365:RNA Helicases