陕北白绒山羊种公羊的体细胞克隆

Somatic cell cloning of Shaanbei white cashmere goat

【目的】利用体细胞核移植技术克隆陕北白绒山羊优秀种公羊个体。【方法】以特别优秀成年陕北白绒山羊种公羊耳部皮肤成纤维细胞为供体细胞,体外培养成熟的陕北白绒山羊卵母细胞作为受体,利用显微操作方法对成熟卵母细胞进行去核操作,然后将供体细胞注射到其卵周隙内,经电融合将其导入去核卵母细胞内形成核移植重组胚。利用钙离子载体Ionomycine联合蛋白酶抑制剂6-甲基氨基嘌呤(6-DMAP)对核移植重组胚进行激活处理,挑选激活后完整的胚胎继续进行体外培养,然后在2～16细胞期时将克隆胚胎移植到相应的同期发情受体母羊体内,利用PCR-RFLP技术鉴定克隆羊。【结果】构建了体细胞核移植胚胎253枚,重组胚胎的融合率为71.54%(181/253),体外培养后的卵裂率为68.33%(123/180),囊胚发育率为25.20%(31/123);在此基础上,构建了537枚克隆胚,将其中卵裂的359枚分别移植到32只代孕母羊体内,移植60d后,有2只受体母羊确认妊娠,最终1只受体母羊妊娠第4月流产出2只死羔;另1只受体母羊维持到期并顺产体细胞克隆羊1只。经遗传学鉴定,2只流产羊羔与1只存活个体均为体细胞核移植后代。【结论】获得陕北白绒山羊克隆个体,建立了相对完善的陕北白绒山羊克隆技术体系。

【Objective】 The objective of present study was to clone Shaanbei white cashmere goat by somatic cell nuclear transfer.【Method】 Skin fibroblasts from adult male Shaanbei white cashmere goat were used as donor cells,and enucleated IVM oocytes were used as recipient ooplasm.Donor cells were introduced into the perivitelline space of recipient ooplasm by microinjection,subsequent fused into ooplasm by double electric pulse.The reconstructed embryos were cultured in vitro after chemical activation by using ionomycine combined application of 6-DMAP,and then the 2-16 cells stage reconstructed embryos were transferred to the corresponding surrogate female goats.【Result】 253 reconstructed embryos were produced,and fusion rate was 71.54%（181/253）,the cleaved rate was 68.33%（123/180）,and blastocyst development rate was 25.20%（31/123）.Then 537 reconstructed embryos were cultured in vitro and 359 cleaved embryos were transferred to 32 surrogate female goats.2 surrogate were diagnosed of pregnancy in 2 months.Finally 1 surrogate miscarried 2 cloned lambs at the 4th gestation month,and the other surrogate produced 1 cloned goat.【Conclusion】 The present study obtained the first cloned offspring of Shaanbei white cashmere goat,whith could provide a platform for the production of transgenetic goats by somatic nuclear transfer.