Isolation And Cloning Of Embryonic Germ Cells From Chicken Primordial Germ Cells

At primary culture, Chicken primordial germ cells (PGCs) were co-culturedwith the gonadal stromal cells. For subculture the PGCs were plated withmitotically inactive feeder layer cells, such as primary mice embryonicfibroblast (PMEF) cells, primary chicken embryonic fibroblast (PCEF) cells,STO cells, SNL cells. The culture system was composed of DMEM (highglucose) supplemented with 2% chicken serum, 0.1mM β-mercaptoethanol,2mM L-glutamine, 10mM HEPES(PH7.6), 20ng/mL conalbumin, 100U/Mlpenicillin,100μg/mL streptomycin,10ng/mL gentamycin, fetal bovine serumand cytokines. The purpose of this study was to investigate the effect ofcytokines and other factors on the culturing of chicken PGCs, to build asuitable culture system and,finally,to obtain chicken embryonic germ cells(CEG cells). The results were as follows:1.Gonadal primordial germ cells (gPGCs) were prepared by isolatingembryonic gonads of 4.5～7.5-day-old (37.8℃, RH60%, titling at a 60°angle once an hour) and dissociating the gonad tissue in 0.25% trypsin-0.02%EDTA. After the inactivation of 0.25% trypsin-0.02% EDTA with DMEMcontaining 10%FBS, the cells were harvested by centrifugation. The gPGCsisolated from four gonads were seeded in one well of the 24-well culture plateand incubated in a CO2 incubator at 37℃ until the gPGCs had colonized as aprimary colony(approximately 2-3 days). For subculturing,the colonies ofCEG cells were agitated by gentle pipetting with 0.25%trypsin-0.02%EDTAtreatment and harvested from the plate after the inactivation of0.25%trypsin-0.02%EDTA with DMEM containing 10% FBS. These cellswere centrifuged at 200×g for 5 minutes and divided into a fresh 24-well 4山东农业大学硕士研究生论文(2004)plate simultaneously plated with PMEF cells, which were mitoticallyinactivated. The CEG cell colonies were passaged at an interval of 5 to 6 dayson average.The morphology of the CEG cell colonies were slightly different from thatof mouse EG cell colonies:the CEG cells did not pack strongly together insmall nests; It was not difficult to discern the individual component cells.Therefore, the CEG cell colonies could easily be dislodged from the feederlayer cells. But, as described for porcine EG (ES) cells, the morphology of theCEG cells was multi-layered and well delineated. The CEG cell wascomposed of a large nucleus and a relatively small amount of cytoplasm. Thenucleolus was not prominent.2.The growth behavior of CEG cells was observed. After being cultured for12h,the small colonies composed of 10-15 PGCs would appear, and thesecolonies became more obvious after another 12h. Culturing at the second(third) day, some nest-like CEG colonies were observed. Some of the primaryCEG colonies were brown, which maybe due to plenty of hemocytes.Hemocytes decreased distinctly after passaged. The cells in small nests werepurified and became singular CEG cells gradually.3.The density of FBS has influence on isolating and cloning of CEG cells.Respective FBS density designed in this study was: 0%,5%,10%,15%.Theresults indicated: that CEG cells in presence of 10% FBS was easiest to isolateand clone.4.Among the factors that influence CEG cells, the supplemented cytokinesare crucial. As a result, the mixture of mLIF,bFGF,hSCF,IGF-1 and h-IL-11has the greatest positive effect on the growth behavior of CEG cells. Therespective dose of mLIF,bFGF,hSCF,IGF-1 and h-IL-11 was: 1000IU/mL,10ng/mL,5ng/mL,10ng/mL,0.04ng/mL. CEG cells were continuouslycultured for 7.0 passages when we appended these five cytokines; In contrast,they were cultured for 2 passages without any cytokine.5.At primary culture, the manner of chicken PGCs co-cultured with the 5王娟:从原始生殖细胞中分离克隆鸡胚胎生殖细胞gonadal stromal cells was the most effective. For subculturing,we usedmitotically inactive feeder layer cells, such as PMEF cells,PCEF cells,STOcells and SNL cells, a...