【作者】 王维；

【导师】 张运海； 章孝荣；

【作者基本信息】 安徽农业大学 ， 动物遗传育种与繁殖， 2010， 硕士

【摘要】 由于在解剖、生理功能上与人类非常相近,猪被认为是人类医学研究的理想模型。猪的体细胞克隆在畜牧业、医学及生物学基础研究上具有重要价值。但是,目前由于效率低下,技术操作繁琐,猪的体细胞克隆还未能直接造福于人类,究其根源主要在于对克隆机理缺乏了解。因此,为进一步优化猪的体细胞克隆平台,建立简单、有效、经济的猪胚胎体外生产体系和探索猪克隆胚早期发育期间DNA甲基化重编程。本研究针对影响猪体细胞核移植中的部分关键环节,如激活和体外培养条件,尝试使用表观修饰剂处理克隆胚提高克隆胚胎发育能力,同时,还针对猪胚胎全基因组DNA甲基化免疫荧光染色检测技术进行优化,跟踪猪克隆胚早期发育的DNA甲基化重编程动态。试验一:研究了200nM的表观修饰剂丙戊酸(VPA)用表观修饰剂对处理克隆胚36h,发现克隆胚的卵裂率和囊胚率有提高的趋势,但差异不显著。试验二:研究了在胚胎培养基中添加肌醇对孤雌胚、克隆胚发育能力的影响,与对照组相比,添加肌醇可提高孤雌胚的囊胚率(76.1% vs 42.7%,P<0.05),但囊胚总细胞数没有增加。对于克隆胚,添加肌醇对克隆胚的囊胚率和囊胚总细胞数上均无显著提高作用,囊胚率(27.8 vs 30.9,P>0.05)。试验三:检验了不同激活方案对孤雌胚和克隆胚发育能力的影响,由此对卵母细胞孤雌与克隆的激活方案进行筛选。本研究用四种激活方案,①电激活后使用CHX处理4h,②电激活前使用10μg/mL CHX中处理10min,③电激活前使用10μg/mL CHX中处理10min后平衡1h再进行电激活,④单独电激活对孤雌胚进行激活,发现这四种方案在卵裂率和囊胚率上没有显著差异。对于克隆胚,本研究使用四种激活方案:①电激活后使用CHX处理4h,②电激活前使用10μg/mL CHX中处理10min,③电激活后使用10μg/mL CHX中处理10min,④单独电激活。结果发现,激活后处理4h和激活前处理10min都显著高于其他两组(P<0.05)。本研究接着针对胎儿成纤维细胞和耳成纤维细胞,用电激活前使用方案②10μg/mL CHX中处理10min和方案④电激活后使用10μg/mL CHX中处理4h两种方案进行激活,发现激活方案②不但节省激活时间,还有比方案④高出接近一倍的囊胚率,是一种简便、稳定、较高效的激活方案。试验四:建立DNA甲基化染色程序,并进行优化,结果发现:使用1:200稀释度的一抗,1:200稀释度的二抗,0.5% TritonX透化30min,能获得更好的免疫荧光染色效果。试验五:使用50nM TSA处理克隆胚30-35h可以显著提高克隆胚的发育能力,在优化的激活方案中,使用TSA处理处理不同品种的猪的体细胞核移植克隆胚,可以提高六白猪耳成纤维细胞和梅山猪胎儿成纤维细胞的克隆胚胎发育能力。试验六:经过对克隆胚的DNA甲基化染色的半定量分析发现,TSA处理可以将克隆胚的DNA甲基化水平降低一倍,但还未达差异显著水平,这表明TSA减少克隆胚胎DNA甲基化水平可能是提高克隆效率的重要原因。综上所述,在猪体细胞克隆中,表观修饰剂VPA处理有提高克隆胚的发育能力趋向,肌醇对孤雌胚胎和克隆胚的发育能力有益,使用50nM TSA处理克隆胚30-35h可以显著提高克隆胚的发育能力,CHX处理重构胚后再电激活10min是简便、高效的激活方案下,在此方案下使用TSA处理克隆胚胎36h,可以提高克隆胚胎发育能力, TSA处理可以将克隆囊胚的DNA甲基化水平降低至一半水平,这可能是提高克隆胚胎发育的能力的原因。更多还原

【Abstract】 Pigs are increasingly regarded as ideal model for human medicine due to anatomical and physiological similarity between pigs and mankind. Somatic cloning in pigs is still limited inside labotoray because of low efficiency, high cost and demanding skills. In orde to optimize and simplify pig cloning procedures, in this study, some key aspects such as activation, in vitro culture of porcine reconstructed embryos were exmined, and some key points engaged in protocols of indirect immuno fluorescence staining against whole genome DNA methylation were also modified prior to examine the DNA methylation reprogramming dynamics in preimplantational embryos in pigs. The experiments were as following.Experiment 1. Effect of valproic acid (VPA), a histone deacetylase inhibitor, treatment after nuclear transfer on in vitro development of pig cloned embryos was investigated. No significant difference between VPA treatment and the control group was found in the cleavage rate (86.67﹪vs. 85.00﹪, P>0.05), or the blastocyst rate (21.33﹪vs. 13.00﹪,P>0.05). The results indicated that addition of 200μM VPA to embryo culture medium for 36 h immediately after activation is not necessary for pig cloned embryos.Experiment 2. The effect of myoinositol (Myo) addition into PZM3 on development of porcine parthenogenetic activated (PA) and somatic cloned embryos. For PA embryos, the blastocyst formation rate (76.1% vs 42.7%,P<0.05) was enhanced by Myo addition; while for SCNT embryos, no difference between the treatment and the control group was observed (27.8 vs 30.9,P>0.05). The results demostrate that embryos from cloning and parthnogentic activation prefere to different nutrient requirement.Experiment 3. In the present study, in order to explore the possibilties of simplify activation manipulations engaged in somatic cloning, the reconstructed oocytes were divided randomly into four groups: electrical activation followed by exposure to cycloheximide (CHX, 10μg/mL) for 4h (ELE+CHX4h, traditional activation), exposure to 10μg/mL CHX for 10min prior to electrical activation (CHX10min+ELE), exposure to 10μg/mL CHX for 10min after electrical activation (ELE+CHX10min), and electrical activation alone (ELE). For PA embryos, similar cleavage rate, blastocyst rate were observed among the four groups. For cloned embryos, however, we found that the CHX10min+ELE group significantly more effective than the ELE group and ELE+CHX10min group, while resulted in similar blastocyst rate to the traditional group. These results suggested that CHX10min+ELE treatment could repace the traditional activation methods without compromizing preimplantation development of cloned embryos, and is an efficient, stable, and simple protocol for activating cloned embryos.Experiment 4. The present experiment was designed to establish and optimize procedures of the indirect immunofluorecence staining against whole genome DNA methylation in porcine PA embryos and cloned embryos. Dilution ratio of primary antibody, and choice of secondary antibodies were selected. The results reveal that if the domestic or imported secondary antibody diluted by the ratio of 1:200, and the primary antibody diluted at 1:300 or 1:200, and the embryos permeabilized by 0.5% Triton-X 100 for 30min, staining of the whole genome DNA methylation will be significantly improved.Experiment 5. Trichostatin A (TSA) is an inhibitor of histone deacetylase, potentially enhancing cloning efficiency. The aim of the present study was to establish the optimal TSA treatment time in order to examine the effect of TSA combined with CHX10min+ELE activation protocol on the cloned embryos. The blastocyst percentage of cloned embryos treated with 50 nM TSA for 30-35 h immediately after activation climbed up to 33.6% (control group: 17.0%; P<0.05). We found that treatment with 50 nM TSA for 36 h after activation notably increased the blastocyst rate compared to the control group. Our data demonstrate that TSA treatment could significantly improve pig nuclear transfer embryos produced by a simple activation method combined with TSA treatment.Experiment 6. TSA-mediated reduction in DNA methyaltion was found by immunofluorescence analysis. Blastocyst stage embryos derived from TSA treatment displayed DNA hypomethylation compared to control embryos, although no significant differences were observed (P>0.05). The results suggest that high blastocyst formation is associated closely with low DNA methylation level, although the exact contribution of inner cell mass or trophoblast should be figure out in future studies.Taken together, the protocol for in vitro production of pig cloned embryos was simplified and improved by using CHX10min+ELE and TSA treatment, and the immunofluorescence staining against 5MeC was improved, which finally help us found that DNA methylation is postively correlated with improved preimplantation development.更多还原