Migration Of In Vitro-Cultured Chicken Primordial Germ Cells In Early Embryo

As the precursors of gametes, primordial germ cells (PGCs) are responsible for delivering genetic information from one generation to the next. Chicken PGCs could be isolated from early embryo, expanded in vitro and genetically modified, then transplanted into recipient embryos to produce germline chimeras. Thus, PGCs is an ideal tool to generate transgene animal. However, production of transgene chimeras was in an inefficient process due to limited availability of knowledge and poor optimization of the migration and colonization of PGCs to gonad. The objective of this study was to explore the factors which affect the efficiency of the migration of chicken PGCs in the recipent embryos, and thus provide basis to optimize the procedure to produce transgenic chickens.Firstly, PGCs were isolated from circulating blood of Guangxi yellow feather chicken embryos at stage 13-15 and then cultured in vitro. After genetically modification and screening by puromycin, a stable PGCs line expressing green fluorescent protein (GFP) was established. Immunocytochemistry revealed that after in vitro culture and genetically modification, these PGCs were still positive for SSEA-1. RT-PCR analysis showed that PGCs express the Dazl, Cvh, PouV, Cdh and Nanog genes. These results demostrated that these GFP-PGCs lines retained their germ cell related properties.Then we investigated the optimum time window of PGCs migration by injecting of these cells into receipient embryo incubated for 50h,55h,60h or 65h. The results revealed that a higher migration efficiency than other groups was observed in embryos incubated for 50h. And the longer incubation of reciepient embryos, the lower migration efficiency was obtained. To find out whether the transplanted cell number would affect migration efficiency,1000, 3000,10000 or 30000 GFP-PGCs were injected to embryos after incubation for 50h. The result shows that the optimum number of PGCs for transplantation was 10,000 per embryo. The migrated PGCs increase as we increasing the injected cell number when the transplant number under 10,000, but the increasing trend wasn’t seemed when over 10,000 PGCs were transplanted. Embryos were hateched after PGCs transplantation and gonads were isolated from a 4-day-old chick. Imaging under fluorescent microscope showed that significant GFP-PGCs colonized the gonads, and PCR detection of chimera chicken sperm DNA showed one of four assumed chimeras is GFP-positive, suggesting successful production of germline chimera.Finally, we try to explore the role of CXCR4 signaling pathway in PGCs migration process. RT-PCR showed that PGCs expressed chemokine receptor 4(CXCR4). Prior to PGC transplantation,0 nM, lOnM,50nM AMD3100 and OnM, lnM,2nM WZ811 were added to cell suspension and then transferred to embryonic vasculature. The result revealed that, supplementation of 50nM AMD3100 and 1nM,2nM WZ811 could significantly inhibit PGCs migration (P<0.05. These results indicated that CXCR4/CXCL12 signaling axis mediated PGCs migration in erly embryo.In summary, Guangxi yellow feather chicken (YF chicken) PGCs can be long-term cultured in vitro and still retained their germ cell related properties after genetically modification. The optimum time window of reciepient embryos for PGCs transplantation is at stage 14, which is corresponding to 50h incubation. And the opitimum PGCs number for transplantation is 10000 per embryo. SDF-1/CXCR4 signaling axis is involved in PGCs migration in early chicken embryos.