2次克隆转基因山羊的重构胚发育分析  

Analysis on the development of reconstituted embryos of secondary cloned transgenic goats

作　　者：宋绍征 陆睿 张婷[2] 潘生强 成勇[2] 周鸣鸣 SONG Shao-zheng;LU Rui;ZHANG Ting;PAN Sheng qiang;CHENG Yong;ZHOU Ming-ming(College of Nursing,Wuxi Taihu University,Wuxi,Jiangsu 214000,China;Jiangsu Provincial Research Center for Animal Transgenesis and Biopharming,College of Veterinary Medicine,Yangzhou University,Yangzhou,Jiangsu 225009,China;School of Pharmaceutical Engineering,Jiangsu Food and Pharmaceutical Science College,Huai'an,Jiangsu 223003,China)

机构地区：[1]无锡太湖学院护理学院,江苏无锡214000 [2]扬州大学兽医学院江苏省转基因动物制药工程研究中心,江苏扬州225009 [3]江苏食品药品职业技术学院,制药工程学院,江苏淮安223003

出　　处：《中国兽医学报》2020年第12期2444-2450,共7页Chinese Journal of Veterinary Science

基　　金：江苏省高校自然科学基金资助项目(19KJB180030);国家转基因生物新品种培育重大专项基金资助项目(2014ZX08008-004);无锡市科协软科学研究重点课题资助项目(KX19B28)。

摘　　要：为了提高体细胞核移植生产转基因克隆山羊的效率,本研究针对2次克隆转基因山羊的重构胚发育情况进行了比较分析。首先,剖宫产从35日龄奶山羊胎儿中分离培养原代胎儿成纤维细胞,分别电转染BLYZ和Blac乳腺特异性表达载体,500mg/L G418筛选、PCR检测以获得转基因细胞株。然后,以此转基因细胞株作为供核细胞用于山羊体细胞核移植。最后,剖宫产获取转基因胎儿克隆细胞系,以此作为供核细胞进行2次克隆生产转基因山羊,并比较分析重构胚发育情况。结果显示:共获得74株LYZ转基因细胞,62株hα-LA转基因细胞;通过第1轮体细胞核移植后,BLYZ和Blac载体的重构卵融合率分别为79.4%(235/296),77.0%(221/287),2~4细胞发育率分别为35.7%(84/235)和33.0%(73/221),移植受体妊娠率分别为38.5%(5/13)和36.4%(4/11);2次克隆后,BLYZ和Blac载体的重构卵融合率分别为90.1%(118/131),87.6%(127/145),2~4细胞发育率分别为66.9%(79/118)和68.5%(87/127),移植受体妊娠率分别为58.3%(7/12)和61.5%(8/13)。以上结果表明,2次克隆的转基因山羊重构胚融合率、发育率和移植受体妊娠率均明显高于传统的体细胞核移植,这为今后高效生产转基因山羊和转基因动物的遗传育种奠定基础。In order to improve the efficiency of the production of transgenic cloned goats by somatic cell nuclear transfer(SCNT),we compared and analyzed the development of reconstructed embryos of secondary cloned transgenic goats.Firstly,the primary fetal fibroblasts were isolated from 35day-old goat fetuses by cesarean section and cultured.Mammary gland-specific expression vectors BLYZ and Blac were electrotransfected into the primary fetal fibroblasts,respectively.The transgenic cells were screened by 500 mg/L G418 and identified by PCR.Then,the transgenic cells were used as donor cells for goat somatic cell nuclear transfer.Finally,the cloned fetal cell lines were obtained by cesarean section and were used as donor cells for secondary cloning to produce transgenic goats,and the development of reconstructed embryos was compared and analyzed.In this study,we obtained 74 LYZ transgenic cell strains and 62 hα-LA transgenic cell strains.After the first round of somatic cell nuclear transfer,the fusion rates of BLYZ and Blac were 79.4%(235/296),77.0%(221/287),respectively,and the development rates of 2-4 cells were 35.7%(84/235),33.0%(73/221),respectively,and the pregnancy rates of the recipients were 38.5%(5/13)and 36.4%(4/11),respectively.After the second cloning,the fusion rate of BLYZ and Blac was 90.1%(118/131)and 87.6%(127/145)respectively,and the development rate of 2-4 cells was 66.9%(79/118)and 68.5%(87/127),respectively,and the pregnancy rate of transplant recipients was 58.3%(7/12)and 61.5%(8/13),respectively.The above results showed that the fusion rate,the development rate and the pregnancy rate of the reconstructed embryos of the secondary cloned transgenic goats were significantly higher than those of the traditional somatic cell nuclear transfer,which laid a foundation for the efficient production of transgenic goats and genetic breeding of transgenic animals in the future.

关 键 词：体细胞核移植 转基因动物 2次克隆 重构胚 山羊 

分 类 号：S826[农业科学—畜牧学]