【作者】 黄雷；

【导师】 李奎；

【作者基本信息】 中国农业科学院 ， 动物遗传育种与繁殖， 2011， 硕士

【摘要】 本文所用的卵巢采集自北京市顺义区鹏程食品有限公司屠宰车间,于2-3小时运送回实验室,运输温度为30-35℃。卵母细胞培养42-44小时后收集成熟卵母细胞用于孤雌激活实验。试验旨在摸索猪卵母细胞孤雌激活的电场强度和脉冲时间,并探索渗透压阶段培养法对孤雌胚胎后期发育的影响。猪卵母细胞成熟培养42-44 h后,分别在电场强度2.1 kv/mm、2.3 kv/mm、2.5 kv/mm和脉冲时间30 ms、60 ms、90 ms的9组电激活参数下进行孤雌激活试验;卵母细胞在2.1 kv/mm和30 ms的参数下进行孤雌激活后,分别培养于渗透压为271 mOsm、280 mOsm、290 mOsm、302 mOsm的PZM-3中,48 h后移入渗透压280 mOsm的PZM-3中继续培养96 h;孤雌胚胎于电激活后先在含2 mmol/L 6-DMAP的PZM-3中培养4-6 h,然后移入不含6-DMAP的PZM-3中继续培养。试验结果表明电场强度和脉冲时间两个参数间无显著的交互作用(P>0.05),三个电场强度试验组间卵裂率无显著差(P>0.05),且30 ms、60 ms、90 ms脉冲时间试验组的卵裂率呈现递减趋势,各试验组的囊胚率无显著差异(P>0.05);孤雌胚胎在渗透压为290-310 mOsm的PZM-3中培养48 h,卵裂率得到显著提高(P<0.05),渗透压对囊胚率无显著影响(P>0.05);6-DMAP对孤雌胚胎卵裂率无显著影响(P>0.05),但可以显著提高囊胚率(P<0.05)。结果提示猪卵母细胞孤雌激活需要较高的电场强度(2.1-2.3 kv/mm)而脉冲时间不宜过长(30 ms);48 h的高渗培养和6-DMAP的辅助激活有助于孤雌胚胎的后期发育。应用红色荧光基因(RFP)标记五指山猪胎儿成纤维细胞,作为猪体细胞克隆技术平台的检测信号,仅仅通过荧光激发就能识别是否是转基因猪,而不通过繁琐的分子生物学检测技术而直观检测,且在胚胎制作过程中可以实时观察制作过程的技术及方法是否可行,对于转基因猪制备有意义。本项研究采用五指山猪近交系的胎儿成纤维细胞,利用Lipofectamine? 2000脂质体转染试剂盒将RFP转化到猪胎儿成纤维细胞,细胞转化阳性率27.52%(79/287),达到本项研究的要求,两种阳性细胞克隆筛选方法均能满足下一步研究所需。试验证实本研究获得的转RFP基因的五指山猪胎儿成纤维细胞是可用的,所采用的技术路线及方法是科学的、可靠的。在RFP细胞筛选过程中,我们利用普通非转基因五指山猪胎儿成纤维细胞和转EGFP基因胎儿成纤维细胞(中科院赵建国老师慧赠)进行了体细胞克隆实验,通过融合率、卵裂率、囊胚率这几个指标对技术平台的各个环节进行逐一调整和优化,提高了单个卵巢卵母细胞的平均获得率(20-23枚),减少了显微操作时间(7-8h),由此提高了体细胞克隆的效率(卵裂率50%,囊胚率12%-15%),并同时开展了移植实验,目前已经移植四头母猪,后续实验正在进行中,移植效率有待进一步检测。更多还原

【Abstract】 Ovaries used in this thesis are collected from the slaughter hall of Pengcheng food company Ltd in Shunyi district of Beijing. The ovaries were carried to laboratory within two to three hours in the temperature environment of 30-35℃. The oocytes were cultured for 42-44h and then collected for parthenogenetic activation. The experiment was conduced to grope the electrical field strength and pulse width of porcine oocyte parthenogenetic and the effect of stage-culture method of osmotic pressure on parthenogentic embryo development. Afer 42-44 h of in vitro maturation, porcine oocytes were activated under the condition of 2.1 kv/mm、2.3 kv/mm、2.5 kv/mm and 30 ms、60 ms、90 ms. Using the parameter of 2.1 kv/mm and 30 ms for parthenogenetic activation and then the oocytes were cultured in PZM-3 with the osmotic pressure of 271 mOsm、280 mOsm、290 mOsm、302 mOsm each other. Afer electrical activation, the oocyte were cultured in PZM-3 with 2 mmol/L 6-DMAP for 4-6 h and then cultured in PZM-3 without 6-DMAP for the last time. The results showed that 1)there were no interaction between electrical field strength and pulse width(P>0.05). Both of the three electrical field strength have no significant effect on cleavage rate(P>0.05). The effect of pulse strengh of 30 ms、60 ms、90 ms on cleavage rate took on the tendency of descending. However, the blastocyte rate of each test group had no significant difference(P>0.05); 2)Cultured for 48 h under the condition of osmotic pressure of 290-310 mOsm in PZM-3,the cleavage rate of parthenogentic embryos were significant higher than other groups(P<0.05), however, there were no significant difference of blastocyte rate(P>0.05); 3)6-DMAP has no significant effect on the cleavage rate of parthenogentic embryo(P>0.05) but the blastocyte rate was significantly increased(P<0.05). These results indicated that porcine oocyte activation needs high electrical field strength(2.1-2.3 kv/mm) but the pulse width must be short(30 ms) ; Hypertonic culture for 48 h and second activation with 6-DMAP will promote the later stage development of parthenogentic embryo.Red fluorescence protein(RFP) gene was used to mark WZSP foetus fibroblast for detection signal of technology platform of porcine somatic clone. We can distinguish the transgentic pig form none just utilizing fluorescence excitation dispensing with molecular biology detection. Otherwise, in the process of embryo production it ensures the real time observation. So that we can evaluate the feasibility of technology and method. It is very important for production of transgentic pig. In this article we have used foetus fibroblast from inbred strais of WZSP. The Lipofectamine? 2000 lipidosome transfection kit was introduced to transfer RFP to porcine foetus fibroblast. The positive rate of cell transfection is 27.52%(79/287) achieving the demand of this research. Both of positive cell clone screening methods are sufficient for next research. Our study has verified that RFP transgenic WZSP foetus fibroblast obtaind in this research is usable and the technology process and method is scientific and reliable. During the time of RFP cell screening we used normal none transgenic WZSP foetus fibroblast and EGFP transgenic foetus fibroblast(Zhao jianguo offering,Chinese Academy of Science) to clone. Three index of fusion rate, cleavage rate and blastocyte rate were used for adjusting and optimizing each section of technology platform. The average acquisition rate of each ovary has been elevated and the period of micromanipulation has been decreased. And then the efficiency of somatic cell clone has been improved(cleavage rate ,50% ; blastocyte rate ,12%-15%). We proceeded with the transplant experiment simultaneously. Up to now we have transplant four sows and follow-up experiment is in progress. The detection of transplant efficiency is pending.更多还原