【作者】 黄永业；

【导师】 欧阳红生；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2014， 博士

【摘要】 体细胞核移植（somatic cell nuclear transfer，SCNT）可以快速大量复制具有优良数量、质量性状家畜个体，是一项有着广泛应用前景重要胚胎工程技术。本实验以提高猪SCNT效率为中心，从三个方面对猪SCNT重编程、胚胎发育影响因素及其分子细胞机制进行研究：（1）分析猪SCNT影响因素；（2）优化猪SCNT胚胎培养体系并研究胚胎发育相关机制；（3）制备嵌合体猪。对作者所在课题组2008年至2012年猪胚胎移植数据统计分析发现：春季进行胚胎移植成功率远高于冬季（33.6%vs.18.6%，p=0.006）；胚胎移植时未排卵代孕母猪怀孕成功率高于已排卵受体母猪（32.0%vs.17.5%，p=0.004）；流产主要集中出现在胚胎移植后第27天到34天之间；根据生存分析结果，可以推测正常受精母猪和代孕母猪分别在117天和125天前产仔完毕；代孕母猪怀孕周期与产仔数呈负相关关系。为了提高克隆效率，使用维生素C、丙戊酸钠（valproic acid，VPA）和Scriptaid对猪SCNT胚胎进行处理，并对相应分子细胞机制进行探索。50μg/ml维生素C处理猪SCNT胚胎15小时，囊胚形成率显著高于对照组（36.0%vs.11.5%, p<0.05）。实时定量检测结果显示，维生素C处理后，囊胚Oct4、Sox2和Klf4表达水平上调。与对照组相比，1mM VPA处理14-16小时可以显著提高SCNT胚胎囊胚形成率（31.8%vs.11.4%, p<0.05）以及克隆效率：VPA处理组中14头受体母猪怀孕（14/20，70%），克隆效率为0.40%；对照组中，6头受体母猪怀孕（6/18，33%），克隆效率为0.18%。与未处理对照胚胎相比，500nM Scriptaid处理SCNT胚胎15小时能促进SCNT胚胎体外囊胚形成（12.2%vs.27.7%, p<0.05）；Scriptaid处理胚胎在二细胞、四细胞和囊胚阶段acH3K14乙酰化水平上升。分别以表达EGFP的雄性长白猪胎儿成纤维细胞和表达tdTomato的雌性松辽黑猪胎儿成纤维细胞作为SCNT供体细胞，并用1mMVPA对重构胚胎进行处理。胚胎发育至四细胞阶段时，对这两种SCNT胚胎以2:2比例进行聚合。聚合胚胎发育到囊胚阶段后进行胚胎移植，成功获得具有明显毛色嵌合特征的克隆猪。更多还原

【Abstract】 Utilizing of Somatic cell nuclear transfer (SCNT) could rapid copy alarge number of animals with excellent quality and quantity traits. Ourresearch was focused on improving porcine SCNT cloning efficiency,uncovering the impacting factors and underlying molecular cellmechanism on porcine SCNT reprogramming and embryonicdevelopment. Three aspects were included in this study: retrospectivestudy on factors related to embryo recipient and embryos transferred;optimizing culturing system for porcine cloned embryos and searchingout the corresponding mechanism; and production of porcine chimeras.The follow-up data related to cloned pig production collected in ourlaboratory was examined. Spring showed a higher full-term pregnancyrate compared with winter. Abortion was most likely to take placebetween Day27to Day34. Non-ovulating surrogate sows presented ahigher percentage of full-term pregnancies compared with ovulating sows.Based on Life Table Survival Analysis, delivery in normally fertilized andsurrogate sows is expected to be completed before Day117or Day125,respectively. Additionally, the length of pregnancy in surrogate sow wasnegatively correlated with the average litter size.To improve porcine cloning efficiency, SCNT embryos were treatedwith vitamin C, histone deacetylase inhibitor Vaproic acid (VPA) andScriptaid. In addition, the relevant molecular and cellular mechanism wasalso determined. Results showed that the blastocyst-formation rate inSCNT embryos treated with50μg/mL vitamin C for15h after activation (36.0%) was significantly higher than that of untreated SCNT embryos(11.5%). The enhanced in vitro development rate of vitamin C-treatedembryos was associated with an higher Oct4, Sox2and Klf4expressionlevels in blastocysts, as determined by real-time PCR. Treatment with1mM VPA for14to16h following activation significantly increased therate of blastocyst formation of porcine SCNT embryos compared to thecontrol (31.8%vs.11.4%). The cloning efficiency in the treated groupwas significantly improved compared to the control group. We found thattreating SCNT embryos with500nM Scriptaid for15h after activationsignificantly enhanced the blastocyst formation rate (27.7%) comparedwith the untreated group (12.2%). It was also found that treating SCNTembryos with Scriptaid increased the level of acH3K14.Chimeric embryos were generated by aggregating two EGFP-cellderived embryos with two tdTomato-cell derived embryos at the4-cellstage. These SCNT embryos were pre-incubated with1mM VPA for15h.After embryo transfer, live piglets with overt coat color chimeras weresuccessfully generated.更多还原