Self renewal, expansion, and transfection of rat spermatogonial stem cells in culture
Self renewal, expansion, and transfection of rat spermatogonial stem cells in culture
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DOI：
10.1073/pnas.0508780102
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发表时间：
2005-11-29
影响因子：
11.1
通讯作者：
Garbers, DL
中科院分区：
文献类型：
--
作者：
Hamra, FK;Chapman, KM;Garbers, DL
使用转基因大鼠，表达增强型绿色荧光蛋白（绿色荧光蛋白）专门在生殖细胞系允许饲养层和污染的睾丸体细胞从生殖细胞和一组精原干细胞标记转录的鉴定分离。以这些分子标记为指导，我们现在已经设计出培养条件，其中大鼠精原干细胞在培养中更新和增殖，倍增时间为3至4天。标记物转录物作为培养时间的函数在相对丰度上增加，并且干细胞在移植到受体大鼠的睾丸后保留定殖和发育成精子细胞的能力。细胞在至少12代后也保持整倍体。细胞系可以被分离和冷冻保存，并且在随后的解冻后，继续自我更新。用含有新霉素磷酸转移酶（neo）选择性标记的质粒转染精原干细胞，导致选择G418抗性细胞系，有效地定殖受体睾丸，这表明基因靶向现在在大鼠中是可行的。
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.