手工克隆生产猪胚胎关键技术的改进研究  

Research on improvement of the key technology of handmade cloning in producing swine embryos

作　　者：陶瑞鑫 贾超[1] 武恩在 李娟[1] TAO Ruixin;JIA Cao;WU Enzai;LI Juan(College of Animal Science and Technology,Nanjing Agricultural University,Nanjing 210095,China;Huai'an NAU New Village Development and Research Co.,Ltd.,Huai'an 223005,China)

机构地区：[1]南京农业大学动物科技学院,江苏南京210095 [2]淮安市南农大新农村发展研究有限公司,江苏淮安223005

出　　处：《畜牧与兽医》2021年第7期13-19,共7页Animal Husbandry & Veterinary Medicine

基　　金：南京农业大学-新疆农业大学联合基金项目(KYYJ201907);江苏省自然科学基金面上项目(BK20171372)。

摘　　要：手工克隆(handmade cloning,HMC)是在传统克隆技术的基础上发展出的一种体细胞核移植技术,作为纯手工操作技术,每一步骤的操作细节都影响着HMC的效率及重构胚胎发育能力。本试验针对HMC生产克隆猪胚胎的3个关键步骤:透明带消化、手工切割去核与无透明带重构胚的体外培养展开研究。结果显示,猪卵母细胞透明带以3.3 mg/mL链酶蛋白酶适度消化(10~15 s),在切割去核时卵母细胞死亡率显著低于透明带的过度(高于15 s)消化,死亡率分别为(3.22±1.08)%和(31.45±2.10)%;当透明带消化时间不足(少于10 s),不能被切割去核的猪卵母细胞显著升高,不可切割率分别为(18.25±4.22)%和(0.57±0.40)%。另外,"铡刀式"操作更适用于手工切割去核,其操作要点对成功获得无核半卵及其胞质含量非常重要。HMC无透明带重构胚体外培养时,孔深约250μm、孔径约300μm的微孔技术(well of wells,WOWs)有利于克隆胚胎发育。本研究优化了HMC操作的关键步骤,为提高生产克隆猪胚胎效率奠定了基础。Cloned mammals have been produced by somatic cell nuclear transfer(SCNT)in different species.However,the SCNT technology cannot be widely applied because of its low efficiency.With the development of the biotechnology,the handmade cloning technology(HMC)has become a novel method that eliminates the complicated operation and expensive micromanipulator.As a artificial technique,the handling tips should be given special attention in the HMC process.Therefore,the present study was designed to investigate the detailed points of HMC,including digestion of zona pellucida(ZP),bisection for enucleation and in vitro culture of ZP-free cloned embryos.The results showed that the death rate of the oocytes from the moderate digestion group(10~15 s)was significantly lower than that from the over digestion group(more than 15 s),(3.22±1.08)%vs(31.45±2.10)%;while the uncuttable rate from the light digestion group(less than 10 s)was significantly higher than that from the moderate digestion,(18.25±4.22)%vs(0.57±0.40)%.The successful enucleation and proper quality of cytoplasm could be achieved by handling the blade correctly.And the well of well system(WOWs)was adopted to culture the zona free reconstructed embryo(about 250μm deep and 300μm in diameter of each micro hole).

关 键 词：体细胞核移植技术 手工克隆 透明带 切割去核 

分 类 号：S828[农业科学—畜牧学]