A Preliminary Study On The Isolation,Culture And Genetic Modification Of Primordial Germ Cells From Yellow Feather Chicken

As precursors of sperms and eggs,Aain primordial germ cells(PGCs)are foreseen as promising tools for generation of transgenic chickens and preservation of avain genetic resources of endangered species.For these purposes,an in vitro culture system enabling avain PGCs to propagate efficiently is necessary.Yellow feather chicken is the most important broiler chicken and widely seen in poultry industry in Guangxi and other provinces in south China.For a better preservation and ulitization of yellow feather chicken,we try to isolate primordial germ cells(PGCs)from yellow feather chicken and establish an in vitro culture system for these cells and investigate the possibility of genetic modification in yellow feather chicken using this cell tool.The PGCs derived from circulating blood(cPGCs)or gonads(gPGCs)of chicken embryos were cultured in direct contact with either irradiated mouse embryonic fibroblasts(MEF)or buffalo rat liver(BRL)cells or cultured in cell culture inserts in the presence of BRL feeder cells.The results showed that the PGCs cultivated by way of cell culture inserts proliferated more rapidly than those in direct contact with BRL or MEF feeders,Overall,33%(1/3)of embryonic blood samples and gonadal samples gave rise to PGCs lines when cultured in cell inserts in the presence of BRL feeder cells,irrespective of the origin from bloods or gonads.All the resulting lines appeared to be lack of female cells,although the initial cells were pooled from male and female embryos,suggesting that male PGCs proliferate more rapidly than female PGCs.The PGCs could proliferate for a extended time in vitro and maintain positive for alkaline phosphatase(AP)staining.Immunocytochemical staining revealed that they expressed germ cell related marker SSEA-1 and EMA-1 and reverse transcription polymerase chain reaction showed that Nanog,PouV,CVH,CDH and DAZL were highly expressed in these cells.The PKH26 labelled PGCs were capable of colonizing the genital ridges of recipient embryos even after cultured in vitro for two months.When these PGCs were transfected with GFP gene constructed in a transposal plasmid with the help of lipofectamin LTX&PLUS,8.5%of PGCs were positive for GFP and a stable GFP line was established after screened in the presence of puromycin.These GFP-PGCs were able to migrate and colonize the gonads after transferred to the bloodstream of stages 13-15 embryos,indicating the germ cell identity even after prolonged culture and genetic modification.In conclusion,we successful isolated PGCs from yellow feather chicken and established a system for long-term culure of these cells in vitro.These PGCs proliferate efficiently in vitro,express all the tested germ cells specific markers,and are capable of colonizing embryonic gonads even after genetically modified.Thus,the PGCs derived in the current study would be a useful cell tool for genetic preservation and modification in avian species.