The Culture Of Chicken Embryonic Primordial Germ Cells In Vitro And Preparation Of Chicken Chimeras By Transfer Of PGCs And Their Molecular Identification By AFLP

Transgenic technology are used for many purposes including enhancing production, creating new breeds, producing products with biological activities. Animal transgenesis is very difficult comparing with plants because of their complicated structure. But bird transgenesis is even harder because the fertilized eggs of birds are large and contain much yolk and are very difficult to handle. Primordial germ cells(PGCs) have, therefore, been considered as a substitute for fertilized eggs for production of transgenic birds. The PGCs are more convenient to manipulate and they can finally differentiate to either spermatogonia in males or oogonia in females.According to the transferred routes in embryos------entering the developing bloodvessels and circulating in the blood stream, finally migrating into the germinal ridge, the PGCs can turn into the vectors to receive foreign DNA for producing transgenic chickens.In this experiment, blood were pooled from chicken embryos hatched for 51-53 hours from the dorsal aorta using a fine glass micropipette wih 40-60 μm diameter. PGCs circulating in blood were purified by Ficoll density gradient centrifugation. The purity of PGCs could be up to 75% by identification based on morphology and PAS (periodic acid-schiff). The PGCs were cultured on stroma cells derived from 5-day-old germinal ridge in Medium 199 supplemented with 10% FBS, 10ng/ml human IGF-1, 10ng/ml bovine FGF-b, and 10U/ml murine LIEThe Guangdong Shiqiza chicken were used as the donor and the Holand H series chicken were used as the recipients. The frozen-thawn PGCs from the donor embryos were isolated and then concentrated up to 400/μl. About 1-1.5μl PGCs suspension were microinjected into the blood vessels of recipient embryos incubated for 60-64 hours. Then these recipient embryos were put back for incubation. Tissues of sexglands from incubated embryos were analyzed by AFLP for identification of germline chimeras. The results showed that 13 of 36 recipients are germline chimeras. The hatched chicks were feeble and dead after their hatching. 8 of them were also analyzed with AFLP to check germline chimeras.To sum up, abundant donor cells are acquired by culturing PGCs in vitro. Refrigeration and preservation of PGCs can be used as strain protection of local chickens,in particular,some endangered genetic resources.