Self Renewal, Expansion, and Transfection of Rat Spermatogonial Stem Cells in Culture

The use of a transgenic line of rats that express enhanced GFP (EGFP) exclusively in the germ line has allowed a separation of feeder layers and contaminating testis somatic cells from germ cells and the identification of a set of spermatogonial stem cell marker transcripts. With these molecular mar...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2005-11, Vol.102 (48), p.17430-17435
Main Authors: Hamra, F. Kent, Chapman, Karen M., Derek M. Nguyen, Ashley A. Williams-Stephens, Hammer, Robert E., Garbers, David L.
Format: Article
Language:English
Subjects:
Animals
Biological Sciences
Cell culture
Cell Culture Techniques
Cell lines
Cell Proliferation
Cultured cells
Feeder cells
Flow Cytometry
Gene expression
Gentamicins
Germ cells
Green Fluorescent Proteins - metabolism
Kanamycin Kinase - genetics
Kanamycin Kinase - metabolism
Male
Medical research
Plasmids - genetics
Rats
Rodents
Sertoli cells
Somatic cells
Spermatogonia
Spermatogonia - cytology
Spermatogonia - metabolism
Stem cells
Stem Cells - cytology
Stem Cells - metabolism
Testes
Testis - cytology
Transfection - methods
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Summary:The use of a transgenic line of rats that express enhanced GFP (EGFP) exclusively in the germ line has allowed a separation of feeder layers and contaminating testis somatic cells from germ cells and the identification of a set of spermatogonial stem cell marker transcripts. With these molecular markers as a guide, we have now devised culture conditions where rat spermatogonial stem cells renew and proliferate in culture with a doubling time between 3 and 4 days. The marker transcripts increase in relative abundance as a function of time in culture, and the stem cells retain competency to colonize and develop into spermatids after transplantation to the testes of recipient rats. The cells also remain euploid after at least 12 passages. Cell lines could be isolated and cryo-preserved and, upon subsequent thawing, continue to self renew. Transfection of the spermatogonial stem cells with a plasmid containing the neomycin phosphotransferase (neo) selectable marker resulted in selection of G418-resistant cell lines that effectively colonize recipient testes, suggesting that gene targeting is now feasible in the rat.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0508780102