Isolating mitotic and meiotic germ cells from male mice by developmental synchronization, staging, and sorting
Name
342832v1.full.pdf
Description
Submitted version
Size
9.85 MB
Format
Adobe PDF
Checksum (MD5)
132392f49f5b52da00eed8332b4ef9ca
Author(s)
Romer, Katherine A.
de Rooij, Dirk G.
Page, David C.
Date Issued
August 2018
Journal
Developmental biology
Publisher
Elsevier BV
Citation
Romer, Katherine A., Dirk G. de Rooij and David C. Page. “Isolating mitotic and meiotic germ cells from male mice by developmental synchronization, staging, and sorting.” Developmental biology 443 (2018): 19-34 © 2018 The Author(s)
Version
Original manuscript
Abstract
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 ~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.
MIT Department
Whitehead Institute for Biomedical Research
Massachusetts Institute of Technology. Computational and Systems Biology Program
Massachusetts Institute of Technology. Department of Biology
Terms of Use
Creative Commons Attribution-NonCommercial-NoDerivs License
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DOI of Published Version
https://doi.org/10.1016/J.YDBIO.2018.08.009
