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Isolation of germline cells from Drosophila embryos by flow cytometry
Author(s) -
Shigenobu Shuji,
Arita Kayo,
Kitadate Yu,
Noda Chiyo,
Kobayashi Satoru
Publication year - 2006
Publication title -
development, growth and differentiation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.864
H-Index - 66
eISSN - 1440-169X
pISSN - 0012-1592
DOI - 10.1111/j.1440-169x.2006.00845.x
Subject(s) - germline , flow cytometry , drosophila (subgenus) , biology , embryo , microbiology and biotechnology , isolation (microbiology) , genetics , gene , bioinformatics
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 .