| Interspecies nuclear transfer has been shown to be a powerful tool for the developmental biology study and rescue of endangered species. Oocytes and embryo of the rabbit (oryctolagus ciniculus) were used to investigate the effect of the cytoskeleton inhibitors on the oocytes parthenogenesis, determine the time of establishment of the cellular gap junction between blastomeres of rabbit embryos and the relationship between this time and the embryo qualities, compare the effects of the two ways of enuclear on the development of the reconstructed interspecies embryo and the developmental potential of the interspecies reconstructed embryos. We also investigated the developmental potential of embryos reconstructed by transferring several kinds of donor cells, which were not treated in quiescence, into enucleated rabbit oocytes. Mediating by liposome, donor cells expressed green fluorescence protein (GFP) were obtained and transferred into recipients to study expression of GFP in preimplantation interspecies reconstructed embryos.On the whole, factors affecting nuclear transplantation have been studied firstly and then the interspecies nuclear transplantation systematically through interspecies nuclear transfer and transgenic interspecies nuclear transfer have been established. The results were as follows:1. Rabbit oocytes were treated with cytoskeleton inhibitor for 1-1.5h and activated by electropulse. No significant effects on the parthenogenesis of the rabbit were found if the oocyte were treated with cytochalacin B (7.5 ug/mL). The developmental rate of 2 -cell (82.2%) and blastocyst (43.3%) were not significantly different from that of the control (76.4% and 51.5%, respectively)( P>0.05). The developmental rate of 2-cell and blastocyst were decreased (64.9% and 23.8% respectively) when the oocytes were treated with colchicines(1.0ug/mL) for 1-1.5 h, significantly different from that of the control (p<0.05). The results from cytochalasin B and plus cochicine treatment were similar to that from treated with cochicines alone. (62.5% 2-cell rate and 30.9% blastocyst rate). Observing through immunofluorescent staining and laser scanning confocal microscopy, we did not found any abnormal of microtubules and microfilaments in blastocysts, derived from oocytes treatment with cytoskeleton inhibitor and parthenogenetic activation.2. Interspecies reconstructed embryos were obtained by transferring and electrofusing nonquiescent giant panda, bovine, mouse somatic cells or embryo stem (ES) cells into enucleated rabbit oocytes, separately. It was shown that these reconstructed embryos could develop into blastocyst stage in vitro, and the highest electrofusion rate (71.3%) was obtained when the ES cells were used as the donors. This electrofusion rate was significantly higher than that of bovine ear fibroblasts (58.5%), mouse granulosa cells (54.8%) (P<0.05), and giant panda fibroblasts (61.8%). When the ES cells were transferred into enucleated rabbit oocytes, 13.8% of the reconstructed eggs developed to blastocysts significantly different from other donor cell groups (P<0.05). This studyindicated that the highest blastocyst rate could be obtained when using ES cells as donor cells, the lowest blastocyst rate was obtained when using mouse somatic cells, and giant panda and bovine somatic cells resulted in similar blastocyst rates.3.The pEGFP-C1 plasmid DNA was transferred to the fibroblasts of giant panda with the help of lipofectamine. It was found that the green fluorescent protein (GFP) could transiently express in fibroblasts after 24-48 hr and stablly express after screening by G418, and the optimum screen concentration of G418 was 400ug/mL. Then we studied the situation of transfection by flow cytometry, and found the optimum pEGFP'cl plasmid DNA concentration and optimum lipsome density, inducing the expression of GFP in 37.4% transfected fibroblasts of giant panda were 3ug/ml and 10 ul/ml respectively. This condition was significant different from others.4. The pEGFP-cl plasmid DNA was tr...
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