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| Separation, Purification and Identification of Primordial Germ Cells from Chicken(Gallus gallus) Embryonic Blood |
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Abstract Avian primordial germ cells (PGCs), as precursor of sperm and eggs, can develop to sperms and eggs in the sexually matured avian. Based on this property, PGCs can be used in the fields of PGCs preservation, transgenic avian preparation and developmental biology and will play very important role in the fields of scientific research and practical application. In this study, the PGCs from blood of chicken (Gallus gallus)embryos which had been hatched to Hamburger-Hamilton stage 13~15 (HH13~15) were separated and purified by Nycodenz density gradient centrifugation. The purity of the PGCs was to 99% and the diameter of the purified PGCs was about 13~15 μm after manually selected by microinjection glass needle. The purified PGCs were identified by alkaline phosphatase(AKP) staining, periodic acid-schiff(PAS) staining and stage-specific embryonic antigens-1(SSEA-1) immunohistochemical staining to show the specific reaction of PGCs. The purified PGCs were labeled with fluorescent dye (PKH 26) and successfully labeled PGCs showed red fluorescence under fluorescent microscope. About 200 labeled PGCs were microinjected into the dorsal aorta of recipient chicken embryos which had hatched to HH14~16. After 8 days of incubation, the gonads of recipient chicken embryos were then separated under a stereoscope and embedded in Neg-50 frozen section medium and frozen in liquid nitrogen. Frozen sections were prepared and observed under fluorescence microscope, the labeled donor PGCs could be observed in the gonad of the recipient chicken embryos, which indicated that the purified PGCs were capable of migrating and settling in gonads of the recipient embryos. This study will lay a foundation for the study of PGCs based germplasm preservation and transgenic technologies.
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Received: 03 January 2014
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