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 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.
Hamra
F Kent
FK
The Cecil H. and Ida Green Center for Reproductive Biology Sciences, Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9051, USA. kent.hamra@utsouthwestern.edu
Chapman
Karen M
KM
Nguyen
Derek M
DM
Williams-Stephens
Ashley A
AA
Hammer
Robert E
RE
Garbers
David L
DL
D000818:Animals; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D005434:Flow Cytometry; D005839:Gentamicins; D049452:Green Fluorescent Proteins / Q000378:metabolism; D019868:Kanamycin Kinase / Q000235:genetics / Q000378:metabolism; D008297:Male; D010957:Plasmids / Q000235:genetics; D051381:Rats; D013093:Spermatogonia / Q000166:cytology* / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D013737:Testis / Q000166:cytology; D014162:Transfection / Q000379:methods
D005839:Gentamicins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C010680:antibiotic G 418; D019868:Kanamycin Kinase