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

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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.