| Cloned pigs by somatic cell have great dominance and potential as models for human disease and and as xenotransplantation, therefore this research has been a hotspot in the field of somatic cell nuclear transfer(SCNT). Up to date, the efficiency of cloned piglets production has been very low, with less than 1-2% of transferred embryos surviving to term. This research focused on establishing a more efficient system for producing pigs by SCNT. Systematical study had been carried on technical factors affecting on efficiency of SCNT by optimizing NT technique, culture conditions. Our aim is to produce enough cloned embryos, which were transferred to surrogate gilts or sows.Fetal fibroblast cell lines were set up from fetuses of Guangxi Bama Xiang-pigs at 40d of gestation and adult ear skin fibroblast cell lines was set up by using explant-seeding method. Pig oviduct epithelium cell lines were also established by scraping and cumulus cells from follicular fluid. And effects of freezing and thawing on the fetal fibroblast cells, cell grow curve and chromosome analysis were discussed. Results showed that freezing and thawing didn’t influence the fetal fibroblast cells proliferation, cell grow curve submitted typical S,92% ploidy of one cell lines maintained normal when cultured up to passage 10.We investigated the parameters for enhanced green fluorescent protein (EGFP) transfected by liposome Liperfectamin 2000 such as the dose of DNA and liposome, the cell confluence and the exposure time of the cell to the DNA-liposome complexes. It was indicated that EGFP expression rate was highest as using 1.0μg DNA,2.4μL liposome and 4h of exposure time as cells covered 85%～90% of the 24-well dish.Cell cycle stage was synchronized by either serum starvation or contact inhibition. Blastocyst rates and cleavage rates in three groups had no significant difference, but blastocyst rates of serum starvation was a little higher than the other two groups.The system of oocytes maturation in vitro (IVM) had been optimized. For oocytes IVM, the culture medium NCSU-23 seemed a little superior than medium TCM199. The maturation rate of oocytes cultured in the NCSU-23 with ten percents of porcine follicular fluid (PFF)was highest (79.08%), and fetal bovine serum (FBS) didn’t help for the oocytes maturation;NCSU-23 medium with or without epidermal growth factor (EGF) had no much difference.Repeated parthenogenetic activation experiments confirmed that in the different activation conditions,200V/mm、60μs、1DC was suitable to improve cleavage rate and blastocyst rate. After electrical activation, the oocytes cultured in the medium with 2mM 6-DMAP were evidently superior in subsequent development. The experiment evaluated the effects of ionomycin concentration and treatment time on parthenogenetic activation, and the result showed that 20μM/L ionomycin treating 40min resulted in the most blastocyst. Oocytes cultured in vitro (IVC) for 44h and 48h profited parthenogenetic activation, meanwhile, the highest cleavage rate for 48h was 81.75% and the highest blastocyst rate for 44h was 22.22%.Compared with McGrath-Solter method, the enucleation rate of spindle-view (95.76%) seemed safer and more efficient. Fetal fibroblast cells as donor cells were fit for development of reconstructed embryos.The fetal fibroblast cell before passage 10 had more advantages in reconstructed embryos development as donor cells than after passage 10. B2 medium was more adequate than NCSU-23 for reconstructed cells activated electrically to develop, but the difference between them was not significant. Considering the experiment cost, NCSU-23 medium was feasible.Embryo transfer was undertook in the 12 Shanghai White Pigs with these reconstructed embryos. One sow succeeded building the pregency and maintained to the terminal. A health Bama mini-pig was borned by the Caesarean birth. In addition, the other three sows were re-estrus until 51d,76d and 77d., respectively after embryo transfer, and the pregnancy rate was 33.3%. |
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