Isolation And Cloning Of Goat Embryonic Stem Cells

This test select goat to study the factor of effects on isolating and cloning Escells. This will develop establishing of goat ES and goat EG lone. The following is result: 1. Primodial germ cells were isolated and cultured from gonads or genital ridges of 46 fetus with age of 20~55days of gestation. Goat EG cell culture medium contains DMEM (high glucose), 15% NBS (or FBS), 0.1 mM ?-mercaptoethanol, 0.01 mM non-essential amino acid, 2 mM glutamine, 100 IU/ml penicillin, and 100 IU/ml streptomycin. One EG cell line had maintained an undifferentiated state for 6 passages. 30～36 days of gestation embryos are optimum for the isolation and culture of goat EG cells. It is difficult to isolate and culture goat EG cells if the age of fetuses is more than 6weeks of gestation. 2. Goat sertoli cells were isolated from goat testicle. Contracted PGCs growing on feeder of GSCs,MEF,GEF,it was found that GSCs>MEF>GEF. The DMEM supplement with 10% LIF is better on cloning goat EG cells. 3. 83 outgrowthing ICMs were obtained from 192 embryos of goat using whole method. ICMs were plated on mitomicin-inactivated MEF feeders and cultured in a humidified environment of 5% CO2 in air, 37 ℃. ES-like colonies were observed and one ES-like cell line had maintained undifferentiation for 8 passages.using 0.125% trypsin+0.02EDTA to digest. The method used mouse ES culture medium composed of DMEM (high glucose) containing 15% FBS, 0.1 mM ?-mercaptoethanol, 0.01 mM non-essential amino acid, 10 ng/ml LIF, 100 IU/ml penicillin, and 100 IU/ml streptomycin. 4. There is no obvious different rate of attaching between the two feeders of MEF and GEF.(P>0.05) . they can promore growth of ES cells in vitro.with the development of goat ,the rate of attaching and multilplication was increasing. The rate of attaching and multiplication of hatched blastocyts is the highest, it was 90%and 60% respectly.the DMEM supplemented with 10% LIF is better on cloning goat ES cells. 5. Goat ES cells were not sensitive to trypsin, using 0.125% trypsin+0.02EDTA and"sereral times dissociation method"was the better way ,bescause this was relative gentle to ES cells and is better for forming new ES clones. 6. The goat ES and EG cells which were pluipotential cells have been indentified by colony morphology, AKP,SSEA,OCT4 staining, in vitrodifferentiation and karyotryping.