Cloning And Characterization Of Ovine Embryonic Stem-like Cells

Embryonic stem cell could be obtained from inner cell mass of preimplanted blastocyst or from primordial germ cells of the early embryos, with the capacity of unlimited growth and differentiation potential. Embryonic stem cell (ESc) can differentiate into different kinds of cells and organs under proper condition, and therefore has attractive prospect in basic research, transplantation and gene therapy. In current research, the morula was obtained from fertilized ovine oocytes that matured in vitro. mouse embryo fibroblast (MEF) was with the embryonic age of 13.5 days used as feeder layer, isolation and culture ovine primary embryonic stem cell was conducted to investigate the method of setting up ovine embryo stem lines. The results obtained as follows.1. MEF was obtained from mouse embryo at the age of 13.5 days, and feeder layer developed from MEF had normal phenotype and excellent viability.2. On the MEF feeder layer, early ovine embryos were used as experiment material to obtaine nest-like colonies of ES-like cells. Detections of dying reaction of AKP was positive to the reaction. These results indicated that the AKP expressed on the surface of ES cells.3. The ovaries from slaughterhouse was sliced to collect oocytes and the result showed that slicing method could remarkably improve the recovery rate of oocytes, the number of A and B grade oocytes were remarkably increased than using aspiration collection method(p<0.05), however, the number of C grade oocytes obtained by aspiration was obvious higher than that by slicing(p<0.05). In vitro maturation rate of occytes got by slicing was remarkably higher than that got by aspiration. Oocytes collected by these two methods showed no difference in in vitro fertiliazation rate(p>0.05).4. Fetal calve serum play a key role in the process of oocytes maturation. The FCS made up 10% or 15% of the total medium made no remarkable difference in oocytes maturation(p>0.05).5. The rate of oocytes maturation in vitro was remarkably increased by adding luteinizing hormone (LH) and epidermal growth factor (EGF), and EGF promoted the expansion of Cumulus.6. Ovaries were transported at the temperature of 10～15℃,20～25℃and 30～35℃irrespectively in 4h after harvested, the maturation rate of oocytes increased when transportation temperature was higher, while the rate of blastula was not consistent with that of matured oocytes. The results indcated that the feasible temperature for transportion ovary.was 20～25℃.7. Oocytes were fertilized after cultured for 16~18h, 20~22h, 24~26h and 28~30h, irrespectively. The oocytes maturation and blastula rate were the lowest after 16 ~ 18h culture, the rate of oocytes maturation was improved and the rate of blastula was the highest after 20 ~ 22h culture. The maturation rate increased while the rate of blastula decreased after 24h culture. Therefore, the optimum time in oocytes maturation was 20~22h in vitro.