绵羊核移植技术的研究
摘要
体细胞核移植技术对于动物育种、保种和濒危动物保护、科学实验及发育生物学等基础理论的研究均有重要意义。本研究对体细胞克隆技术在绵羊繁育及品种改良方面的应用提供技术支持。对核移植技术的研究发现:组织块培养法与酶消化法相比虽然培养时间较长,但选用组织块培养法可有效防止细胞在原代培养和传代过程中的污染,而且利用较长消化时间可以得到纯绵羊皮肤成纤维细胞系;通过两种发育液的对比发现发情羊血清对卵母细胞的生长有着重要作用,最后选定了发育液B液(15%发情羊血清,10μg/ml FSH,20μg/ml LH,1.5μg/ml 17-?E2的TCM199);在电压1000V/cm~1800V/cm区间对卵母细胞进行物理激活,得出1400V/cm激活电压为最适宜电压,获得卵裂率、桑椹胚率和囊胚率最高。对比物理激活和化学激活两种方法对卵母细胞发育的影响,采用物理激活(1400V/cm)其卵裂率和桑椹/囊胚率分别为70.3%和21.3%;采用离子霉素联合6-DMAP激活处理绵羊卵母细胞,其卵裂率和桑椹\囊胚率分别为80.1%和38.3%;采用7%乙醇联合6-DMAP激活处理绵羊卵母细胞,其卵裂率和桑椹\囊胚率分别为78.3%和35.2%。化学激活与物理激活相比较中发现化学激活法比物理激活更加有效,两种化学激活法中离子霉素联合6-DMAP激活法较7%乙醇联合6-DMAP激活法处理绵羊卵母细胞的卵裂率和桑椹\囊胚率高。在电融合试验中对比了1100 V/cm -1600 V/cm这一区间的融合效果,得出1200 V/cm时融合率最高,当电压升至1400 V/cm时卵裂率可达47.1%,但桑椹胚率不高。最终选定电压1200 V / cm-1300 V/cm为最佳电压。克隆胚胎移植共进行39次,生产体细胞克隆胚290多枚,移植受体母羊39只,2个情期后未返情有10只,对妊娠母羊的护理工作犹为注意,但是依然发生了胎儿流产的情况,目前为止只产生一只羔羊。
Somatic cell nuclear transfer technology is important for animal breeding、conservation and basic theory of protection of endangered species、scientific experiments and developmental biology. This study provides technical support on the application of somatic cell cloning in sheep breeding and variety improvement. Through the nuclear transfer studies ,we have found: compared with enzyme digestion, although it training a longer time, tissue block culture method can prevent cells polluted in process of primary culture and passage, besides,pure sheep skin fibroblast cell line can be obtained using a longer digestion time. Through contrast of two developmental medium we found, estrus sheep serum plays an important role on the growth of the oocyte, developmental medium of liquid B (15% estrus sheep serum, 10μg/ml FSH, 20μg/ml LH, 1.5μg/ml 17-?E2 of TCM199) was finally selected.Made physical activation on oocytes in the voltage range on 1000V/cm ~ 1800V/cm , found 1400V/cm voltage is the most appropriate, access to the cleavage rate, morula rate and blastocyst rate is the highest. In comparison of effect of physical activation and chemical activation on oocyte development, the cleavage rate and morulae / blastocysts ratio were 70.3% and 21.3% by physical activation (1400V/cm); in Ionomycin combined with 6-DMAP activation treatment,the cleavage rate and morulae \ blastocyst ratio were 80.1% and 38.3% ; in 7% ethanol combined with 6-DMAP activation treatment of sheep oocytes, the cleavage rate and morulae \ blastocyst ratio were 78.3% and 35.2%. Compared chemical activation with physical activation, the chemical activation method was found more effective than physical activation, Compared two chemical activation method , ionomycin combined 6-DMAP activation method has higher cleavage and morulae \ blastocyst rate than treatment of 7% ethanol combined 6-DMAP activation . In electrofusion experiments, compared integration effects in the range of the 1100 V / cm -1600 V / cm, obtained the highest fusion rate on 1200 V / cm, when the voltage is increased to 1400 V / cm, the cleavage rate up to 47.1% , but not high of morula. Finally we selected voltage 1200 V / cm -1300 V / cm as the best voltage. cloned embryo transfer was carried a total of 39 times, more than 290 pieces somatic cell cloned embryos were produced, 39 sheep recipients were transplanted , there were 10 sheep did not return to estrus after two sexual period , work on the care of pregnant sheep was paid attention, but fetal abortion still happened, so far produced only one lamb.
Somatic cell nuclear transfer technology is important for animal breeding、conservation and basic theory of protection of endangered species、scientific experiments and developmental biology. This study provides technical support on the application of somatic cell cloning in sheep breeding and variety improvement. Through the nuclear transfer studies ,we have found: compared with enzyme digestion, although it training a longer time, tissue block culture method can prevent cells polluted in process of primary culture and passage, besides,pure sheep skin fibroblast cell line can be obtained using a longer digestion time. Through contrast of two developmental medium we found, estrus sheep serum plays an important role on the growth of the oocyte, developmental medium of liquid B (15% estrus sheep serum, 10μg/ml FSH, 20μg/ml LH, 1.5μg/ml 17-?E2 of TCM199) was finally selected.Made physical activation on oocytes in the voltage range on 1000V/cm ~ 1800V/cm , found 1400V/cm voltage is the most appropriate, access to the cleavage rate, morula rate and blastocyst rate is the highest. In comparison of effect of physical activation and chemical activation on oocyte development, the cleavage rate and morulae / blastocysts ratio were 70.3% and 21.3% by physical activation (1400V/cm); in Ionomycin combined with 6-DMAP activation treatment,the cleavage rate and morulae \ blastocyst ratio were 80.1% and 38.3% ; in 7% ethanol combined with 6-DMAP activation treatment of sheep oocytes, the cleavage rate and morulae \ blastocyst ratio were 78.3% and 35.2%. Compared chemical activation with physical activation, the chemical activation method was found more effective than physical activation, Compared two chemical activation method , ionomycin combined 6-DMAP activation method has higher cleavage and morulae \ blastocyst rate than treatment of 7% ethanol combined 6-DMAP activation . In electrofusion experiments, compared integration effects in the range of the 1100 V / cm -1600 V / cm, obtained the highest fusion rate on 1200 V / cm, when the voltage is increased to 1400 V / cm, the cleavage rate up to 47.1% , but not high of morula. Finally we selected voltage 1200 V / cm -1300 V / cm as the best voltage. cloned embryo transfer was carried a total of 39 times, more than 290 pieces somatic cell cloned embryos were produced, 39 sheep recipients were transplanted , there were 10 sheep did not return to estrus after two sexual period , work on the care of pregnant sheep was paid attention, but fetal abortion still happened, so far produced only one lamb.
引文
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