Isolation of germline cells from Drosophila embryos by flow cytometry.
Primordial germ cells (PGC) are the earliest identifiable germ cells in the embryo. To understand the molecular basis of germline development, isolation of pure PGC is required. We report here the use of fluorescence-activated cell sorting (FACS) to isolate pure populations of Drosophila pole cells, which are the presumptive primordial germ cells in flies. In order to fluorescently mark pole cells, we used an EGFP-vasa transgenic line that expresses green fluorescent protein (GFP) specifically and continuously in the germ line throughout the life cycle. The purity of FACS-sorted pole cells from embryos was confirmed by microscopic inspection and quantitative polymerase chain reaction. Moreover, by optimizing the sample preparation and the sorting protocol, embryonic gonads could also be isolated. This technique opens the way for genome-wide transcriptome analysis of germline cells. In a pilot experiment, we generated a cDNA library from purified embryonic gonad and identified a novel germline-specific gene, RpL22-like.
Shigenobu
Shuji
S
Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, Higashiyama, Myodaiji, Japan.
Arita
Kayo
K
Kitadate
Yu
Y
Noda
Chiyo
C
Kobayashi
Satoru
S
D000818:Animals; D030801:Animals, Genetically Modified; D002460:Cell Line / Q000166:cytology* / Q000502:physiology; D053487:DEAD-box RNA Helicases; D018076:DNA, Complementary / Q000235:genetics; D004330:Drosophila / Q000166:cytology / Q000196:embryology* / Q000235:genetics; D029721:Drosophila Proteins / Q000032:analysis / Q000235:genetics; D004625:Embryo, Nonmammalian / Q000166:cytology; D020224:Expressed Sequence Tags; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D005718:Gametogenesis / Q000235:genetics*; D005854:Germ Cells / Q000737:chemistry / Q000166:cytology / Q000254:growth & development*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D008297:Male; D020365:RNA Helicases / Q000032:analysis / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics; D012269:Ribosomal Proteins / Q000235:genetics
D018076:DNA, Complementary; D029721:Drosophila Proteins; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; C489368:RpL22 protein, Drosophila; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C088269:vas protein, Drosophila; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases