The Preparation And Cultivation Of Porcine Parthenogenetic Tetraploid Embryos In Vitro

The tetraploid cell will present the serious deviation distribution phenomenon in the embryo organization growth process, because it only allocated to the extra-embryonic organization's formation in the individual process. Chimeric tetraploid embryo with the certain amount ES cell or iPS cell, in chimeric embryonic development process, its extra-embryonic organization mainly from tetraploid cell, while the body entirely from the ES cell or the iPS cell, this technology is known as tetraploid compensation technology. In 2009, Zhou Qi etc obtain the live-born iPS mouse by tetraploid complementation, which proven that iPS cells maintain a pluripotent potential that is very close to ES cells for the first time in the world, and provided the solid theoretical foundation for iPS technology application.In 2009, chinese took the lead in the world to generate induced pluripotent stem cells of pig. Theoretically, may obtain the clone porcine completely from iPS cell through the tetraploid compensation technology. This study is focus on explore the pig tetraploid parthenogenetic embryo's preparation method and optimizate condition in vitro. Finally, we detected the tetraploid embryo quality. The experimental results were as follows:1. In the porcine oocyte mature later period, no adding hormone the maturation fluid to obtain the higher rate of maturation compared to the adding hormone mature fluid(68.8% & 60.7%), but no statistically significant difference (P > 0.05);2. In the PZM-3 embryo culture medium with 3 mg/mL BSA group had higher parthenogenetic cleavage rate and blastocyst rate (67.8%; 31.3%);3. In the PZM-3 embryo culture medium with 10% serum the embryo blastocyst rate and hatching blastocyst rate was slightly better than not add group (27.3% & 22.9%; 0.05% & 0.03%), but no significant difference (P > 0.05). The blastocysts of two group stained by Hoechst 33342, observed under fluorescence microscope. Showed that adding serum group blastocyst had more cells and smaller nucleus than do not add serum group;4. Screened the electricity fusion parameter of porcine parthenogenetically 2-cell embryos, resulted that 375/25 x 2(two square DC pulses, electric field was 375 V/mm, pulse duration of 25μs) would obtain relatively better fusion rate (92%) and cleavage rate (62%);5. Expression and location of pluripotent factors were examinede by real-time PCR and the immunity fluorescence staining on tetraploid parthenogenetic blastocysts, result showed that the porcine tetraploid parthenogenetic blastocysts expressed mRNA of pluripotent gene nanog, oct4, sox2, although expression lower than parthenogenetic blastocysts, but the difference was not significant. The immunity fluorescence examined protein expression and localization, found that three genes expressed and localized in the blastocyst cell nucleus;6. Injected iPS cell separately into the perivitelline space of 2-cell-stage,4-cell-stage,6-cell-stage,8-cell-stage and morula tetraploid embryo and tetraploid blastocoele by micromanipulation, there was not chimeric embryo in vitro culture process.