Cloned goats (Capra hircus) from adult ear cells

The average number of available oocytes recovered per ovary collected during the breeding season in dairy goats was 5.5 (1815/330). 66.17% (1201/1815) of oocytes extruded the first polar body after maturation in vitro for 20 h. 75.44% (906/1201) of matured oocytes with membrane evagination around the MII chromosomes were enucleated. Ear skin fibroblast cells were derived from an adult female dining Grey goat (C. hircus). The cells were cryopreserved in liquid nitrogen after passage 2. Thawed cells were further cultured for 3-6 passages and were subjected to serum starvation by 0.5% FBS for 2-10 d, then used as donor cells for nuclear transfer. 98.12% (889/906) of the enucleated oocytes were reconstructed by intracytoplasmic injection of karyoplast. The reconstructed embryos were activated by 5μ mol/L ionomycin for 4.5 min and further activated by culturing with 6-dimethylaminopurine (6-DMAP) for 3 h. After 36 h of culture in mCR1aaBF, 76.69% (645/841) of the cloned embryos cleaved. There were no signifi     

成年耳细胞克隆山羊(Capra hircus)

繁殖季节采集关中奶山羊卵巢,采集获得可用卵母细胞5.5枚/卵巢(1815/330).经约20 h 成熟培养,第一极体排放率66.17%(1201/1815).将有类第二极体排出结构的成熟卵母细胞去核,去核率75.44%(906/1201).培养济宁青山羊耳部皮肤成纤维细胞传2代后液氮冷冻,解冻培养3~6代,用0.5% FBS饥饿2~10 d作为供体细胞.利用卵母细胞胞质内注射法将分离的供体细胞核胞体注入到去核卵母细胞内,注核成功率98.12%(889/906).克隆胚胎用5μmol/L 离子酶素激活4.5 min, 在含2 mmol/L 6-二甲氨基嘌呤(6 dime-thylaminopurine,6-DMAP)的培养液中培养3 h,然后在mCR1aaBF培养液中培养36 h,卵裂率76.69%(645/841).其中由未经冷藏处理供体细胞克隆得到308枚胚胎,激活后卵裂率、4-细胞发育率、囊胚发育率分别为68.5%(211/308),59.72%(126/211)和17.46% (22/126);另外,由4℃冷藏处理24h供体细胞获得的109枚克隆胚胎,激活率、4-细胞率、囊胚率分别为72.48%(79/109),53.16%(42/79)和19.05%(8/42).统计学分析表明,4℃处理供体细胞对克隆胚胎的早期发育没有显著影响.将102枚发育至4-细胞期的克隆胚胎移植到17只自然发情2~3 d 的受体山羊输卵管,其中非冷藏供体细胞克隆的84枚胚胎移植后没有产仔.18枚冷藏体细胞克隆的胚胎移植获得1只发育足月的克隆山羊.将冷藏或非冷藏处理供体细胞克隆得到的19枚体外发育囊胚移植到自然发情6~8 d 的受体山羊,结果无一产仔.未经冷藏处理供体细胞克隆得到的18枚体外发育桑椹胚移植到自然发情5 d受体山羊后获得1只足月羔羊.微卫星引物PCR扩增结果证实2只克隆山羊来源于同一供体细胞.     

Cloned goats (Capra hircus) from adult ear cells

The average number of available oocytes recovered per ovary collected during the breeding season in dairy goats was 5.5 (1815/330). 66.17% (1201/1815) of oocytes extruded the first polar body after maturation in vitro for 20 h. 75.44% (906/1201) of matured oocytes with membrane evagination around the MⅡchromosomes were enucleated. Ear skin fibroblast cells were derived from an adult female Jining Grey goat (C. hircus). The cells were cryopreserved in liquid nitrogen after passage 2. Thawed cells were further cultured for 3-6 passages and were subjected to serum starvation by 0.5% FBS for 2-10 d, then used as donor cells for nuclear transfer. 98.12% (889/906) of the enucleated oocytes were reconstructed by intracytoplasmic injection of karyoplast. The reconstructed embryos were activated by 5 μmol/L ionomycin for 4.5 min and further activated by culturing with 6-dimethylaminopurine (6-DMAP) for 3 h. After 36 h of culture in mCR1aaBF, 76.69% (645/841) of the cloned embryos cleaved. There were no significant differences in development in vitro between the cloned embryos derived from donor cells precooled at 4℃ for 24 h and nonprecooled donor cells. The cleavage rates, 4-cell development, and blastocyst development of reconstructed embryos were 72.48% (79/109), 53.16% (42/79), and 19.05% (8/42) in precooled group; 68.5% (211/308), 59.72% (126/211), and 17.46% (22/126) in nonprecooled group, respectively. Eighteen cloned 4-cell embryos derived from precooled donor cells were transferred and one cloned kid was born. Eighty-four cloned 4-cell embryos derived from nonprecooled donor cells were transferred and no offspring were produced. Of 18 cloned morale from nonprecooled donor cells transferred, one kid was born. The results of microsatellite DNA analyses indicated that the two cloned kids were from the same donor fibroblast cell line derived from an adult goat ear skin.