Self renewal, expansion, and transfection of rat spermatogonial stem cells in culture

Franklin K Hamra, Karen M. Chapman, Derek M. Nguyen, Ashley A. Williams-Stephens, Robert E Hammer, David L. Garbers

Research output: Contribution to journalArticle

165 Scopus citations

Abstract

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 cryopreserved 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.

Original languageEnglish (US)
Pages (from-to)17430-17435
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume102
Issue number48
DOIs
StatePublished - Nov 29 2005

Keywords

  • Enhanced GFT fluorescence
  • Gene targeting
  • Germ line
  • Spermatogenesis

ASJC Scopus subject areas

  • General

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