Study on the methods introducing exogenous DNA into chicken primordial germ cells

本研究旨在探索将外源DNA导入鸡原始生殖细胞并产生转基因鸡的方法。一种从胚胎血细胞中分离原始生殖细胞的方法首次得到发展并得到进一步改进。然后，在体外和体内尝试用脂肪转染和核转染的方法转染分离的原始生殖细胞。结果表明，GFP基因在体外和体内通过脂肪转染的方式被高效导入原始生殖细胞，并在发育中的胚胎性腺中得到了较强的表达。转染法也能有效地将GFP基因导入原始生殖细胞。另一方面，利用线粒体D-loop区域的单核苷酸多态性分析了从胚胎血液中分离的原始生殖细胞转移到受体胚胎的X期囊胚后的命运。证实移植的原始生殖细胞成功迁移到受体胚胎的生发脊上。该方法可应用于利用精子样本分析推测的雌雄嵌合鸡的种系嵌合性。此外，还进行了原始生殖细胞的体外培养。部分原始生殖细胞群成功维持并在饲养细胞上增殖。需要改善培养条件，以保持原始生殖细胞不分化。

The present study was carried out to develop methods to introduce exogenous DNA into chicken primordial germ cells and produce transgenic chickens. A method to isolate primordial germ cells from embryonic blood cells was first developed and further improved. Then, transfection of isolated primordial germ cells was attempted by lipofection and nucleofection in vitro and in vivo. As a result, GFP gene was efficiently introduced into the primordial germ cells by lipofection in vitro and in vivo and the introduced GFP gene was expressed strongly in the gonads of developing embryos. Nucleofection method was also effective introducing GFP gene into primordial germ cells. On the other hand, the fate of primordial germ cells isolated from embryonic blood was analyzed after transfer into stage X blastoderm of recipient embryos using single nucleotide polymorphism present in the mitochondrial D-loop region. It was confirmed that the transferred primordial germ cells were successfully migrated to the germinal ridges of recipient embryos. This method can be applied to analyze the germline chimerism of putative male and female chimeric chickens using sperm samples. Furthermore, in vitro culture of primordial germ cells was carried out. A part of the primordial germ cell population was successfully maintained and proliferated on the feeder cells. It is required to improve culture conditions so as to maintain primordial germ cells undifferentiated for the future.