胚盘下腔显微注射慢病毒载体制备转基因鹌鹑  

Introduction of transgenic quail produced by microinject lentiviral vector into subgerminal cavity

作　　者：张自富[1] 赵瑜[1] 胡静[1] 贺东阳 刘锦妮[1] 李洵[1] 章平[1] 刘纪成[1] 张广强[1] 陈培荣[1] 

机构地区：[1]信阳农林学院动物科学系,河南信阳464000

出　　处：《中国兽医学报》2016年第7期1186-1192,共7页Chinese Journal of Veterinary Science

基　　金：信阳农林学院博士基金项目(201201020);河南省博士后研发基地项目(2014-69);河南省基础与前沿技术研究计划资助项目(B20143690)

摘　　要：利用显微注射法给每枚新鲜鹌鹑种蛋胚盘下腔注射滴度为1×109 TU/mL人类免疫缺陷病毒I型(human immunodeficiency virus-1,HIV-1)慢病毒载体1μL,直接封口后孵化出雏,应用倒置荧光显微镜及分子生物学方法检测。结果显示:在注射慢病毒后第4天,倒置荧光显微镜下观察到发育胚胎卵黄膜上绿色荧光蛋白较强表达;试验操作240枚鹌鹑种蛋,孵化出雏34只,孵化率为14.1%;对新出雏鹌鹑解剖后,可检测到喙部、眼部、羽毛、肝脏、肾脏、盲肠、肺脏、小肠、大肠、输卵管、心脏、胸肌及大脑等内脏器官中绿色荧光蛋白广泛性表达,但在性腺中绿色荧光蛋白表达信号较弱;对发育到性成熟期的G0代19只雄性鹌鹑精液基因组聚合酶链式反应(polymerase chain reaction,PCR)检测,其中3只为阳性,阳性率为15.7%(3/19);在3只阳性雄性鹌鹑的236只后代中,有4只经PCR及印迹杂交(Southern-blot)检测为阳性,阳性率为1.69%(4/236)。结果表明:利用慢病毒载体胚盘下腔注射成功生产出转基因鹌鹑,为转基因禽类制备提供了一种简便易行的好方法。The aim of this study was to explore a new,easy and effective way to produce transgenic birds.We combined the lentiviral vector with microinjection teachonolgy together,and used the parrafilm to seal chicken shells instead of the culture models of substitute egg shells and complicated Ⅲ stage cultivate.1μL of lentiviral vector（human immunodeficiency virus-1,HIV-1）,containing an enhanced-green fluorescent protein（eGFP）,was microinjected into the subgerminal cavity of the fresh fertilized eggs at the titer of 1×109 TU/mL,the egg shells were then directly sealed using parrafilm untill hatch.The embryos and newly hatched quails were then analyzed by fluorescence microscopy and molecular biological methods.After 4days incubation,strong expression of green fluorescence protein can be observed in the vitelline membrane under the fluorescence microscorp.In a total of 240 embryos injected,34 quails（14.1%）were successfully hatched.In newly hatched quails,expression was detected in the beak,eye,feather,liver,kidney,caecum,lung,small intestine,large intestine,oviduct,heart,breast muscle and brain,while the transgene expression in the gonads was greatly reduced or completely absent.Semen samples were collected from the hatched 19 sexually matured male quails and tested by polymerase chain reaction（PCR）,the results showed that transgene was present in three G0 male quails 3/19（15.7%）,and 4/236G1offspring（1.69%）from the 3positive G0 quails were transgenic.It indicated that transgenic quail could be produced by microinject lentiviral vector into subgerminal cavity.

关 键 词：胚盘下腔 显微注射 慢病毒载体 转基因禽类 增强型绿色荧光蛋白 

分 类 号：S813.3[农业科学—畜牧学] S852.65[农业科学—畜牧兽医]