Isolation And Culture Of Goat Primordial Germ Cells

Under certain conditions, Primordial germ cells(PGCs) maintain the undifferentiated state, which can be established to embryonic stem cells (ESCs) lines through long-term culture in vitro, known as embryonic germ cells(EGCs). EGCs is another pluripotent stem cells following the ESCs and embryonal carcinoma cells (ECCs). In this study, The fetal genital ridge from Jiangsu local white goat were as experimental materials, we isolated and cultured EGCs from PGCs, then EGCs were subcultured and identificatied in vitro. Some factors influencing the isolation and culture of goat PGCs had been discussed in this paper, Our fundamental work may be useful to establish and further study in the biology of local white goat ESCs lines.The results were as follows:1. The growth behavior in vitro of three kinds of cells including Mouse embryonic fibroblast cells (MEF),Goat embryonic fibroblast cells (GEF), Mouse Sertoli cells(MSCs) were similar, the time of their adherence was very short and they proliferated rapidly. The proliferation of GEF cells were inhibited after treated with 10μg/mL mytomycin-c for 3 hours, and it was the appropriate duration for mytomycin-c treatment. GEF could be Cryopreserved in -70℃in the short time, and the recovery rates of it could be up to training requirements.2. PGCs were colony-like and nest-like through cultured in vitro, they were raised on fibroblast cells feeder layer. the shape of EGCs were similar to PGCs in vitro culture after passages.3.Co-culture and MEF, GEF were studied as the feeding layers, when the cytokines were not added in the medium, only the primary colons were able to observe; PGCs could be transmitted to the second generation Only by GEF as the feeder layer, primary colons could be observed in the other three cultured methods,when the cytokines were added in the medium. the primary colons were unable to observe by MSCs as the feeder layer, when the cytokines were added or not in the medium .4. Fetal genital ridges were digested 15min at room temperature (25℃) , the results of 0.125% Trypsin +0.02% EDTA gourp was better than 0.25% Trypsin +0.04% EDTA gourp, the Former gourp was in higher primary colony rate, the Significant was difference (p <0.05). Effects of different passage methods on passages of PGCs were studied in this paper, PGCs can be transmitted to the second generation in "picked colony method" or "trypsin digestion method", Two methods was not significant (p> 0.05).5. Different concentrations and types of cytokines were added in the medium,①LIF: 10 ng/ml;②LIF: 20 ng/mL;③LIF (10 ng/mL) + SCF (10 ng/mL);④LIF (20 ng/mL) + SCF (20 ng/mL) , when GEF was as the feeder layer, the results were that the average primary colony rates were not significant (p> 0.05) in the four groups, and the primary colons could be transmitted to the second generation.6.Effects of different gestational age on isolation and passage of PGCs were studied, only a primary colon was observed in 4 fetus of crown-rump length less than 15 mm, and no primary colon was observed in 6 fetus of crown-rump length more than 30 mm.7. The second generation of EGCs were Cryopreserved in liquid nitrogen in this study , the result was that the recovery was not successful, the recovery rate was zero.