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Abstract 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℃ 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.
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Received: 05 September 2011
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