As progenitors of spermatogonia and oogonia, primordial germ cells(PGCs) are characterized by their pluripotency, therefore they represent a good model for the study of embryo development in vitro. Owing to the physiological and developmental characteristics of poultry, PGCs have great value in transgenic research. In our study, PGCs isolated from the genital ridges of 5.5 days' Meiling chicken (Gallus domesticus) embryo were cultured in 24-well plates at 37.5°C in a water-saturated atmosphere of 95% air and 5% CO2 in vitro. The PGCs were stained by histochemical and immunohistochemical for periodic acid-schiff (PAS), alkaline phosphatase (AKP), SSEA-1 (stage-specific embryonic antigens-1) and TERT(telomerase reverse transcriptase). The gene expression of Cvh (chicken vasa homologue), Cdh (chicken dead end homolog) and Dazl (deleted in azoospermia-like), PouV (POU domain class 5 transcription factor 1), Nanog (nanog homeobox) and Sox2 (sex determining region Y-box 2) in the PGCs were analyzed by fluorescence quantitative PCR. The foreign gene of fluorescent protein (pEGFPN3) was transfected into these PGCs by lipofectin method, and the gene transfection efficiency was analyzed by the concentration of plasmid DNA, liposomes and different incubation time. The results showed that chicken PGCs colony with a typical nest-like structure were positive for PAS, AKP, SSEA-1 and TERT staining, and the EG cells could form embryoid bodies. RT-PCR analysis in chicken PGCs showed that genes of stage specific type of PGCs, Cvh, Cdh and Dazl, and pluripotent stem cell-related genes PouV (Oct-4 homologue), Nanog and Sox-2, were expressed significantly than that in the CEF (chicken embryonic fibroblast). In addition, after 24 h transfection, the fluorescence could be observed in cytoplasm and nucleus, and the transfection efficiency of three fluorescent protein genes was up to 16%. This study provides a good material for gene regulation in PGCs differentiation, gene marker and transgenic animals.%作为精原细胞和卵原细胞的祖细胞,由于其全能性的特点可作为体外胚胎发育研究的良好模型,而禽类独特的生理和发育特点使得其原始生殖细胞(PGCs)在转基因研究中有着巨大价值.本研究通过对孵化5.5 d的梅岭土鸡(Galluus domesticus)胚生殖腺中分离得到的PGCs,接种于24孔培养板在温度37℃、95％空气和5％oCO2的培养箱中进行体外培养；借助组织化学及免疫组织化学染色对PAS (高碘酸希夫氏染色)、AKP(碱性磷酸酶染色)、SSEA-1(胚胎阶段特异性表面抗原-1)以及TERT(端粒酶反转录酶)进行了鉴定;采用荧光定量PCR技术对PGCs阶段特异性表达基因Cvh(鸡Vas a基因同系物)、Cdh(鸡死端同系物)、Dazl(类无精症缺失)和干细胞多能性相关基因PouV(POU结构域V类转录因子1)、Nanog(nanog同源异型盒)、Sox-2(性别决定区Y盒2)基因表达进行了分析；利用脂质体介导法,将绿色荧光蛋白(pEGFP-N3)基因导入鸡PGCs细胞,结果表明,PGCs集落有典型的鸟巢状结构,且PAS、AKP、SSEA-1以及TERT染色阳性；PGCs阶段特异性表达基因Cvh、Cdh、Dazl和干细胞多能性相关基因PouV、Nanog 和Sox-2在鸡PGCs中均远远高于在鸡成纤维细胞(CEF)中的表达；转染24 h后,绿色荧光蛋白均匀的充满细胞质和细胞核,转染效率最高为16％,研究结果为禽类PGCs分化过程中基因调控及基因标记、转基因动物克隆等技术提供了良好基础.
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