Culture And Identification Of Goat Embryonic Stem Like Cells And PGCs

Experiments were performed to determine the effect factor of isolation , cloning and passage of embryonic stem cells ( ES) in goat , factors include sourse of embryos , plant methed ,feeder cells ,coat conditions and growth factors.Then culture and identified the goat PGCs cells in vitro .1.According to ICM goat embryos in vivo culture conditions.plant half blastocyst on MEF feeder culture in high glucose and low glucose DMEM DMEM/F12 medium .After the blastocyst attached stripping ICM by glass needle. The results showed: low glucose DMEM more suitable for goats ICM proliferation and colony formation ,in the culture medium 1 ICM adherence rate was 35.7% can be passaged 5 times, the culture medium 2 adherence rate was 26.3% can be passaged 2 times,the difference was significant. Planted half or hatching goat blastocyst on goat fetal fibroblast feeder layer, bovine fetal liver fibroblast feeder layers and primary mouse embryonic fibroblast feeder cells, found that both the species of feeder layer cells and embryonic treatments were significantly affected the attach rate and growth of GES, Half of the goat in vivo developed blastocysts on the MEF feeder posted the highest attach rate 55.6%, can be passaged 6 times in vitro, and on goat fetal fibroblast feeder layer goat ICM get the lowest adherence rate of 10%, can be passaged 2 times in vitro.2.In order to compare the effect of gelatin and ECM to goat ES cell culture .Plant the half of the in vitro developed embryos on the PMEF feeder layer, with culture medium 1 and found that the adherence rate of ICM in gelatin was 35.7%, the adherence rate of ICM in ECM-coated culture dishes was 40%, can be passage 5 times, the difference was not significant.3.This study compared adhesion rate of the in vitro developed blastocyst and clone blastocyst and subculture, result showed that half embryos in the ECM-coated culture dishes, PMEF feeder layer culture medium 1 adherent rate of clone blastocyst was 25%, can be passage 2 times, in vitro development of blastocyst ICM adherence rate was 40%, can be passage 5 times, the difference was Significant.4.Isolation theprimordial germ cells (PGCs) and the surrounding tissue from Inner Mongolia cashmere goat fetus,cultured in three culture systems, results showed that: The A culture system without any factors can only be passaged one generation, B culture system add only LIF and Insulin could be passage four generations, C culture system adding LIF, Insulin and high-quality fetal bovine serum culture system can be passage 9 generations,5. Identified stem like cells and PGCs by RT-PCR, AKP staining, and immunofluorescence.AKP identified was positive, RT-PCR detection ofβ-actin, hTERT, GAPDH, Nanog, Oct3/4, immunofluorescence detection of Oct3/4, SSEA-3, SSEA-4 were positive. In vitro differentiation culture can found nerve cells, fat cells and epithelial cells like cells...