| Cloned pigs by somatic cell have great dominance and potential as models for humandisease and as human xenotransplantation, therefore this research has been a hotspot inthe field of somatic cell nuclear transfer(SCNT). Systematical study had been carded ontechnical factors affecting on efficiency of SCNT by optimizing NT technique, cultureconditions and high-efficiency methods. Our aim is to enhance the efficiency of pigs SCNT.Fetal fibroblast cell lines were set up from fetuses of Guangxi Bama Xiang-pigs at 40dof gestation and adult ear skin fibroblast cell lines was set up by using explant-seedingmethod. We also established pig oviduct epithelium cell lines by scraping and cumulus cellsfrom follicular fluid. Effects of freezing and thawing on the fetal fibroblast ceils, cell growcurve and chromosome analysis were discussed. Results showed that freezing and thawingdidn'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 ,thecell confluence and the exposure time of the cell to the DNA-liposome complexes. It wasindicated that EGFP expression rate was highest as using 1.0μg DNA, 2.4μL liposome and4h 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 teams had no significant difference, butblastocyst rates of serum starvation was a little higher than the other two teams.The system of oocytes maturation in vitro (IVM) had been optimized. For oocytesIVM ,the culture medium NCSU-23 seemed a little superior than medium TCM199.Thematuration rate of oocytes cultured in the NCSU-23 with ten percents of porcine follicularfluid (PFF)was highest (79.08％), and fetal bovine serum (FBS) didn't help for the oocytesmaturation; NCSU-23 medium with or without epidermal growth factor (EGF) had nomuch difference.Repeated parthenogenetic activation experiments confirmed that in the differentactivation conditions, 200V/mm, 60μs,1DC was suitable to improve cleavage rate andblastocyst rate. After electrical activation, the oocytes cultured in the medium with 2mM 6-DMAP were evidently superior in subsequent development. The experiment evaluatedthe effects of ionomycin concentration and treatment time on parthenogenetic activation,and as the result, 20μM/L ionomycin treating 40min obtained the most blastocyst. Oocytescultured in vitro (IVC) for 44h and 48h profited parthenogenetic activation, meanwhile, thehighest cleavage rate for 48h was 81.75% and the highest blastocyst rate for 44h was22.22%.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 fordevelopment of reconstructed embryos.The fetal fibroblast cell before passage10 had more advantages in reconstructedembryos development as donor cells than after passage 10. B2 medium was more adequatethan NCSU-23 for reconstructed cells activated electrically to develop, but the differencebetween them was not significant. Considering the experiment cost, NCSU-23 medium wasfeasible. |
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