期刊
DEVELOPMENTAL BIOLOGY
卷 443, 期 1, 页码 19-34出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ydbio.2018.08.009
关键词
Germ cells; Testis; Spermatogenesis; Meiosis; Synchronization; Epigenomics
资金
- Howard Hughes Medical Institute
- NIH Pre-Doctoral Training Grant [T32GM007287]
Isolating discrete populations of germ cells from the mouse testis is challenging, because the adult testis contains germ cells at every step of spermatogenesis, in addition to somatic cells. We present a novel method for isolating precise, high-purity populations of male germ cells. We first synchronize germ cell development in vivo by manipulating retinoic acid metabolism, and perform histological staging to verify synchronization. We use fluorescence-activated cell sorting to separate the synchronized differentiating germ cells from contaminating somatic cells and undifferentiated spermatogonia. We achieve similar to 90% purity at each step of development from undifferentiated spermatogonia through late meiotic prophase. Utilizing this 3 S method (synchronize, stage, and sort), we can separate germ cell types that were previously challenging or impossible to distinguish, with sufficient yield for epigenetic and biochemical studies. 3 S expands the toolkit of germ cell sorting methods, and should facilitate detailed characterization of molecular and biochemical changes that occur during the mitotic and meiotic phases of spermatogenesis.
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