慢病毒载体介导的血管显微注射法制备转基因鸡的研究  

Study on the effect of preparing transgenic chiken by microinjection of lentiviral vector

作　　者：张自富[1] 赵瑜[1] 胡静[1] 秦清明 麻冰洁 赵聘[1] 陈思睿[2] 赵云焕[1] ZHANG Zifu;ZHAO Yu;HU Jing;QIN Qingming;MA Bingjie;ZHAO Pin;CHEN Sirui;ZHAO Yunhuan(College of Animal Science and Technology,Xinyang College of Agricultural and Forestry,Xinyang 464000,China;National Engineering Laboratory for animal Breeding,College of Animal Science and Technology,China Agricultural University,Beijing100193,China)

机构地区：[1]信阳农林学院动物科技学院,河南信阳464000 [2]中国农业大学动物科技学院畜禽育种国家工程实验室,北京100193

出　　处：《中国兽医科学》2025年第3期415-420,共6页Chinese Veterinary Science

基　　金：河南省自然科学基金项目(182300410028);信阳农林学院高水平科研孵化器建设基金项目(FCL202104);大别山区家禽种质资源应用与健康养殖科技创新团队项目(XNKJTD-013)。

摘　　要：由于禽类独特的生殖生理特点和胚胎发育的复杂性,笔者利用慢病毒载体与鸡胚血管显微注射法相结合,以期探寻出一种简便、有效的制备转基因鸡的方法。对发育至第14~15期鸡胚,于卵黄外周静脉血管显微注射1μL病毒滴度为1×10^(9)TU/mL携带由人源性泛素启动子C调控的eGFP慢病毒,试验共操作了76枚胚胎,出雏44只,孵化率为57.9%(44/76)。结果显示,经检测,在G0代雏鸡的喙部、眼部、羽毛、大脑、胸肌、肺、腺胃、肠、脾、肝、心脏、肾中均观测到绿色荧光蛋白广泛性表达;在随机抽检G0代7只小鸡的性腺中,均观测到绿色荧光蛋白超强表达100%(7/7);对发育到性成熟期的母鸡和公鸡随机抽样解剖,观测到睾丸和卵巢的卵泡中绿色荧光蛋白广泛性表达;对G1代60只雏鸡经PCR检测发现14只为阳性,阳性率为23.3%(14/60),Southern-blot检测结果进一步证实为阳性。结果表明,利用该方法成功生产出转基因鸡,为家禽基因组编辑创立了一种方便快捷、高效稳定的新方法。In order to identify the availability of early embryo blood vessels microinjection of lentiviral vector as an new,effective way in making transgenic chicken,transgenic technology as microinjection and lentiviral vector were combined according to the characteristics of chicken development.1μL lentiviral vector,containing an enhanced-green fluorescent protein gene(e GFP)regulated by of Human ubiquitin C(h UBC) promoter,was microinjected into the vitelline peripheral venous vessels of chicken embryos at Hamburger-Hamilton stage 14-15(HH14-15)at the titer of 1×10^(9)TU/m L.A total of 76embryos were injected and 44 chicken(44/76,57.9%) were successfully hatched.In newly hatched G0 chicks,e GFP shown as the presence of green fluorescence was widely expressed in the beak,eye,feather,brain,pectoralis,lung,glandular stomach,intestine,spleen,liver,heart and kidney,especially in gonads.After chicks raised to sexual maturity,cockerels and hens were dissected by random sampling,and e GFP expression was detected in testis and follicle.60 G1 offspring was detected by polymerase chain reaction(PCR),14 were e GFP-positive(14/60,23.3%),Southern-blot and genetic analyses revealed the same result.It indicated that infection of PGCs with lentiviral vector via direct injection into blood vessels has the potential to provide a more convenient and efficient way to produce transgenic poultry.

关 键 词：转基因鸡 慢病毒载体 显微注射 绿色荧光蛋白 

分 类 号：S831.2[农业科学—畜牧学]