马体细胞核移植的研究
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摘要
体细胞核移植技术为家畜快速繁育和基因组保存提供了新的机遇和方法。在我国,这项技术在牛羊等家畜的繁育上已得到应用,但在马的繁殖上还很少开展这方面的研究。本研究以纯血马的体细胞为供核细胞,分别以马和驴的卵母细胞为胞质受体构建同种和异种体细胞核移植胚,建立了马的体细胞培养体系及马和驴卵母细胞的体外成熟培养体系,并对胚胎的体外培养、手术法移植早期异构克隆胚到受体马输卵管进行了相关的研究。
1.马体细胞系的建立
本研究通过对马腿部皮肤组织的体外培养、冻存和复苏的实验研究,建立了马成纤维细胞的培养和保存体系。
2.马体细胞核移植胚的构建
通过对马卵母细胞两种采集方法的对比,发现切割法比抽吸法更适合马卵母细胞的收集,可获得较高的回收效率。以M199和DMEM/F12为基础培养液用于马卵母细胞的体外成熟培养,结果显示成熟率差异不显著。扩展型(Ex) COCs体外培养24-28小时和紧凑型(Cp) COCs体外培养30-36小时后成熟率分别达到65.2%和42.9%,两种类型COCs成熟后的MⅡ期卵母细胞且具有相同的发育能力。马体细胞核移植实验中发现140V,20Us, 2次脉冲的条件可获得较高的融合效率,融合率达72.5%。采用Ionomycin与6-DMAP和CHX联合激活,重构配的卵裂率为41.9%。
3.马-驴异种核移植胚的构建
本实验分别采用M199培养液,DMEM/F12培养液和由M199培养液与DMEM/F12培养液按1:1混合组成的培养液作为驴卵母细胞的体外成熟基础培养液,最终成熟率差异不显著(p>0.05),并且这三种成熟液驴卵母细胞的孤雌激活和发育无显著影响。提高成熟液中钙离子的浓度不能有效提高驴卵母细胞体外成熟和孤雌激活的效率。驴的COCs同马的COCs相似也分为扩展型(Ex) COCs和紧凑型(Cp) COCs,两种类型的COCs分别成熟培养30-36小时和24-28小时后获得MⅡ期卵母细胞的效率分别为75.9%和55.2%。驴卵母细胞在室温下放置于EH培养液中12小时内对卵母细胞的体外成熟率无显著影响。长时间(20-22小时)的运输卵巢、卵母细胞或是使用简易便携式培养系统在运输途中成熟培养驴卵母细胞都不利于卵母细胞的成熟,但后两种方法的成熟效率高于前一种方法。SOFaa和DMEM/F12都可用于驴的胚胎体外培养,差异不显著(p>0.05)。提高发育液中葡萄糖的浓度可促进胚胎的发育,但差异不显著。以马皮肤成纤维细胞和去核的驴卵母细胞胞质体构建马-驴异种核移植胚胎,在融合参数为130-150V,20Us,2次脉冲时可获得较高的融合率,融合率为60-70%,采用Ionomycin与6-DMAP和CHX联合激活,重构配的卵裂率为48.4%。共移植了46枚早期马-驴异种核移植胚胎到5匹受体马的输卵管中,90天检测均未建立妊娠。
The technique of somatic cell nuclear transfer provides a new method and opportunities for fast breeding and genome preservation in domestic animals. In China, this technology has been applied in some domestic animals, such as cattle and sheep. However, there are fewer researches for horse reproduction on this area. In this study, we reconstruct somatic cell nuclear transfer embryo by fibroblast of thoroughbred horse and equine oocytes, and also reconstruct interspecies somatic cell nuclear transfer embryo by fibroblast of thoroughbred horse and donkey oocytes. We have established in vitro maturation system for equine and donkey oocytes. We also do some work for embryo in vitro culture and transplanted early heterogeneous cloned embryos into recipient oviducts by surgical method.
1. The establishment of equine somatic cell lines
In the study, the suitable culture and preservation system for the horse fibroblasts was set up during the study of culturing the horse leg skin cells in vitro, freezing and thawing the cells.
2. Reconstruction somatic nuclear tranfer embryo of horse
Both of the M199 and DMEM/F12 were used for in vitro maturation and the result show that there have no significant difference (p>0.05).After 24-28h and 30-36h maturation for (Ex) COCs and (Cp) COCs,gained 65% and 43% maturation rate ,respectively. MⅡstage oocyte wether gained from (Ex) COCs or (Cp) COCs have same developmental competence. In the study for equine nuclear transfer ,wo found that the highest fusion rate wound gained(72.5%) when the fusion parameter were 140 V,20 Us, 2 pulse。The cleavage rate of reconstructed embryo is 41.9%.
3. Reconstruction of horse-donkey interspecies somatic nuclear tranfer embryos
Three medium that M199、MEM/F12 and equal parts (v/v) of M199 and DMEM/F12 were evaluated for their ability to support in vitro maturation of donkey oocytes and their development after parthenogenetic activation. There were no significant differences among the three maturation media for oocyte maturation and their development after parthenogenetic activation (p> 0.05). As equine COCs,there also have two types[(Ex) COCs and (Cp) COCs]for donkey COCs. After 24-28h and 30-36h maturation for (Ex) COCs and (Cp) COCs, gained approximate 70% and 50% maturation rate, respectively. Transport donkey ovaries within 6 h were not affect the maturation rate of donkey oocytes. There were no effect for maturation rate of donkey oocytes when them were treated with EH medium for 12h at room temperature. Wether transport donkey ovary or oocyte for a long time(20-22h),or maturation of the oocytes occurs during shipment using a portable incubator were effectless for in vitro maturation of donkey oocytes. Both the SOFaa and DMEM/F12 can used for in vitro culture of donkey embryo, there have no significant difference(p>0.05).DMEM/F12 supplement with high level of glucose (10mM) wound be better for in vitro culture of donkey parthenogenetic embryo, but there have no significant difference . In the study of produced horse - donkey heterogeneous embryo use equine fibroblasts and anucleated donkey oocytes by somatic cell nuclear transfer, we found that the high fusion rate(60-70%) were gained when the fusion parameter were 130-150 V,20 Us, 2 pulse. The cleavage rate of reconstructed embryo is 48.4%. We transplanted 46 early horse - donkey heterogeneous embryos into oviducts of five recipient horses, no pregant were detected after 90 days
1.马体细胞系的建立
本研究通过对马腿部皮肤组织的体外培养、冻存和复苏的实验研究,建立了马成纤维细胞的培养和保存体系。
2.马体细胞核移植胚的构建
通过对马卵母细胞两种采集方法的对比,发现切割法比抽吸法更适合马卵母细胞的收集,可获得较高的回收效率。以M199和DMEM/F12为基础培养液用于马卵母细胞的体外成熟培养,结果显示成熟率差异不显著。扩展型(Ex) COCs体外培养24-28小时和紧凑型(Cp) COCs体外培养30-36小时后成熟率分别达到65.2%和42.9%,两种类型COCs成熟后的MⅡ期卵母细胞且具有相同的发育能力。马体细胞核移植实验中发现140V,20Us, 2次脉冲的条件可获得较高的融合效率,融合率达72.5%。采用Ionomycin与6-DMAP和CHX联合激活,重构配的卵裂率为41.9%。
3.马-驴异种核移植胚的构建
本实验分别采用M199培养液,DMEM/F12培养液和由M199培养液与DMEM/F12培养液按1:1混合组成的培养液作为驴卵母细胞的体外成熟基础培养液,最终成熟率差异不显著(p>0.05),并且这三种成熟液驴卵母细胞的孤雌激活和发育无显著影响。提高成熟液中钙离子的浓度不能有效提高驴卵母细胞体外成熟和孤雌激活的效率。驴的COCs同马的COCs相似也分为扩展型(Ex) COCs和紧凑型(Cp) COCs,两种类型的COCs分别成熟培养30-36小时和24-28小时后获得MⅡ期卵母细胞的效率分别为75.9%和55.2%。驴卵母细胞在室温下放置于EH培养液中12小时内对卵母细胞的体外成熟率无显著影响。长时间(20-22小时)的运输卵巢、卵母细胞或是使用简易便携式培养系统在运输途中成熟培养驴卵母细胞都不利于卵母细胞的成熟,但后两种方法的成熟效率高于前一种方法。SOFaa和DMEM/F12都可用于驴的胚胎体外培养,差异不显著(p>0.05)。提高发育液中葡萄糖的浓度可促进胚胎的发育,但差异不显著。以马皮肤成纤维细胞和去核的驴卵母细胞胞质体构建马-驴异种核移植胚胎,在融合参数为130-150V,20Us,2次脉冲时可获得较高的融合率,融合率为60-70%,采用Ionomycin与6-DMAP和CHX联合激活,重构配的卵裂率为48.4%。共移植了46枚早期马-驴异种核移植胚胎到5匹受体马的输卵管中,90天检测均未建立妊娠。
The technique of somatic cell nuclear transfer provides a new method and opportunities for fast breeding and genome preservation in domestic animals. In China, this technology has been applied in some domestic animals, such as cattle and sheep. However, there are fewer researches for horse reproduction on this area. In this study, we reconstruct somatic cell nuclear transfer embryo by fibroblast of thoroughbred horse and equine oocytes, and also reconstruct interspecies somatic cell nuclear transfer embryo by fibroblast of thoroughbred horse and donkey oocytes. We have established in vitro maturation system for equine and donkey oocytes. We also do some work for embryo in vitro culture and transplanted early heterogeneous cloned embryos into recipient oviducts by surgical method.
1. The establishment of equine somatic cell lines
In the study, the suitable culture and preservation system for the horse fibroblasts was set up during the study of culturing the horse leg skin cells in vitro, freezing and thawing the cells.
2. Reconstruction somatic nuclear tranfer embryo of horse
Both of the M199 and DMEM/F12 were used for in vitro maturation and the result show that there have no significant difference (p>0.05).After 24-28h and 30-36h maturation for (Ex) COCs and (Cp) COCs,gained 65% and 43% maturation rate ,respectively. MⅡstage oocyte wether gained from (Ex) COCs or (Cp) COCs have same developmental competence. In the study for equine nuclear transfer ,wo found that the highest fusion rate wound gained(72.5%) when the fusion parameter were 140 V,20 Us, 2 pulse。The cleavage rate of reconstructed embryo is 41.9%.
3. Reconstruction of horse-donkey interspecies somatic nuclear tranfer embryos
Three medium that M199、MEM/F12 and equal parts (v/v) of M199 and DMEM/F12 were evaluated for their ability to support in vitro maturation of donkey oocytes and their development after parthenogenetic activation. There were no significant differences among the three maturation media for oocyte maturation and their development after parthenogenetic activation (p> 0.05). As equine COCs,there also have two types[(Ex) COCs and (Cp) COCs]for donkey COCs. After 24-28h and 30-36h maturation for (Ex) COCs and (Cp) COCs, gained approximate 70% and 50% maturation rate, respectively. Transport donkey ovaries within 6 h were not affect the maturation rate of donkey oocytes. There were no effect for maturation rate of donkey oocytes when them were treated with EH medium for 12h at room temperature. Wether transport donkey ovary or oocyte for a long time(20-22h),or maturation of the oocytes occurs during shipment using a portable incubator were effectless for in vitro maturation of donkey oocytes. Both the SOFaa and DMEM/F12 can used for in vitro culture of donkey embryo, there have no significant difference(p>0.05).DMEM/F12 supplement with high level of glucose (10mM) wound be better for in vitro culture of donkey parthenogenetic embryo, but there have no significant difference . In the study of produced horse - donkey heterogeneous embryo use equine fibroblasts and anucleated donkey oocytes by somatic cell nuclear transfer, we found that the high fusion rate(60-70%) were gained when the fusion parameter were 130-150 V,20 Us, 2 pulse. The cleavage rate of reconstructed embryo is 48.4%. We transplanted 46 early horse - donkey heterogeneous embryos into oviducts of five recipient horses, no pregant were detected after 90 days
引文
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