卵周隙内注射慢病毒生产转基因绵羊的方法  

Establishment of a technique to generate transgenic sheep by perivitelline injection of lentivirus vector

作　　者：汪立芹[1,2] 刘晨曦[1,2] 王静 黄俊成[1,2] 

机构地区：[1]新疆动物生物技术重点开放实验室,乌鲁木齐830000 [2]农业部草食家畜繁育生物技术重点开放实验室,乌鲁木齐830000

出　　处：《中国实验动物学报》2011年第1期12-15,F0003,共5页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家转基因重大专项(编号:2008ZX08008-003;2008ZX08008-001-3;2008ZX08010-004-008)

摘　　要：目的提高并稳定转基因绵羊生产效率。方法采用卵周隙内注射慢病毒的方法生产转基因绵羊。比较了不同发育阶段卵母细胞注射慢病毒对其后期发育及基因表达的影响,对照了注射不同慢病毒剂量以及不同熟练程度人员操作,对胚胎发育及基因表达率的影响。结果 (1)受精前或受精后注射慢病毒对胚胎后期发育和阳性率没有影响(P>0.05);(2)在受精后的单细胞注射或者2～4细胞期注射慢病毒对胚胎后期发育和阳性率也都没有影响(P>0.05);(3)慢病毒注射剂量在50～100 pL之间对转基因效率没有影响(P>0.05);(4)操作人员的熟练程度不影响胚胎成活率和转基因效率(P>0.05)。结论建立了卵周隙内注射慢病毒生产转基因绵羊的方法,并使转基因胚胎阳性率稳定在70%以上。Objective To explore a technique to improve the efficacy of generation of transgenic sheep by injection of lentivirus into the perivitelline space of oocytes.Methods Recombinant EGFP lentivirus was injected into the perivitelline space of oocytes.The growth status and gene expression of oocytes at different stages of embryonic development were assessed to analyze whether the injection doses of the lentivirus or the skill of different operators influence the embryonic development.Results（1） The lentivirus injection before or after fertilization of oocytes had no significant influence on the embryo development and gene expression（P〉0.05）;（2） After fertilization,the lentivirus injection into single firtilized cell or 2-4-cell embryos did not affect the embryonic development and gene expression;（3） Lentivirus injected in a dose of 50 pL to 100 pL did not affect the transfection efficacy（P〉0.05）;（4） The proficiency of operators did not affect the embryo survival rate and transfection efficacy（P〉0.05）.Conclusions A method of generation of transgenic sheep by injecting lentiviral vector into perivitelline space has been established,with a stable positive rate of transgenic embryos higher than 70%.

关 键 词：转基因 慢病毒 绵羊卵母细胞 

分 类 号：Q95-33[生物学—动物学] R-33[医药卫生]