[1]刘勤,张晋宇,王露露,等.慢病毒载体介导的增强型绿色荧光蛋白转基因小鼠的建立[J].第三军医大学学报,2008,30(10):885-889.
 LIU Qin,ZHANG Jin-yu,WANG Lu-lu,et al.Production of EGFP transgenic mice by lentiviral transgenesis[J].J Third Mil Med Univ,2008,30(10):885-889.
点击复制

慢病毒载体介导的增强型绿色荧光蛋白转基因小鼠的建立(/HTML )
分享到:

《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
30卷
期数:
2008年第10期
页码:
885-889
栏目:
论著
出版日期:
2008-05-30

文章信息/Info

Title:
Production of EGFP transgenic mice by lentiviral transgenesis
作者:
刘勤张晋宇王露露刘昌峨王勇万瑛
第三军医大学基础医学部:动物学教研室,全军免疫学研究所
Author(s):
LIU Qin ZHANG Jin-yu WANG Lu-lu LIU Chang-e WANG Yong WAN Ying
Department of Laboratory Animal Science, Institute of Immunology, College of Medicine, Third Military Medical University
关键词:
慢病毒载体增强型绿色荧光蛋白转基因小鼠
Keywords:
lentiviral vector enhanced green fluorescent protein transgenic mice
分类号:
R-332;R373;R394.1
文献标志码:
A
摘要:
目的  以慢病毒作为载体,制作增强型绿色荧光蛋白(EGFP)转基因小鼠,建立慢病毒介导的转基因动物制备技术平台。  方法  慢病毒包装采用三质粒系统。3个质粒分别为转基因质粒FUGW、病毒结构蛋白表达质粒psPAX2以及病毒包膜蛋白表达质粒pMD2.G。病毒包装时,用磷酸钙沉淀法将三质粒共转染来源于人胚肾细胞系的293FT细胞,培养48 h后,收取含病毒的上清,并通过高速离心浓缩病毒。将含浓缩病毒液作梯度稀释后感染293FT细胞,通过流式细胞计数仪测定病毒滴度。用显微注射法将浓缩的病毒液注射至FVB/N小鼠1-细胞期胚胎透明带下。在荧光显微镜下观察胚胎EGFP的表达。将注射后发育至2-细胞期的胚胎移植至假孕受体母鼠,得F0代小鼠。通过紫外光照射观测EGFP在小鼠活体内的表达水平。  结果  病毒液浓缩前的滴度≥106 TU/ml(transducing unit,TU),经过高速离心对病毒进行浓缩和纯化,其滴度达到109 TU/ml以上。将浓缩病毒液注射至小鼠1-细胞期胚胎透明带下,注射后胚胎的2-细胞期卵裂率为81.8%(1 189/1 453),利用荧光显微镜分别在注射后60、84、132 h观察胚胎,均发现有较强荧光。在2-细胞期,每一视野下胚胎阳性率>90%,说明包装的病毒成功并高效地转染小鼠胚胎。胚胎移植后假孕母鼠妊娠率为42.9%(12/28),首建鼠阳性率为60.8%(73/120),转基因小鼠的总体研制效率(转基因小鼠数/注射胚胎数)为5.0%(73/1 453)。将F0代EGFP转基因小鼠分别与野生型小鼠交配,在其F1、F2、F3代小鼠中均获得了EGFP阳性小鼠,阳性率分别为91.4%(32/35)、93.8%(30/32)、93.1%(27/29)。  结论  通过慢病毒载体感染小鼠1-细胞期胚胎可有效地制备转基因小鼠。我们已初步建立了慢病毒介导的转基因小鼠制备技术体系。
Abstract:
Objective    To establish an efficient and reliable method for transgenic mouse production, we performed lentiviral vector-mediated transgenesis in mice.     Methods    The leniviral vector FUGW was packaged by co-transfection of 293FT cells with two packaging vectors, psPAX2 and pMD2.G. The two packaging vectors provide structural proteins and envelope protein respectively which are necessary for the proper package of pseudotype virus. The tire of the pseudotype virus was tested by FACS after infecting 293FT cells with the solution containing the virus particles. The virus was concentrated by high speed centrifugation, and concentrated virus solution was injected into perivitelline space of mouse 1-cell eggs. EGFP expression in pre-implantation eggs was observed under fluorescent microscope, and that in mouse body under UV light.     Results    The titre of the primary virus solution was ≥106 TU/ml, and that after concentration was ≥109 TU/ml. The concentrated virus solution efficiently infected mouse eggs after injected into the perivitelline space. On 60, 84, 132 h after injection, EGFP expression was observed in the injected eggs. At 24 h after injection when the majority of the injected eggs was in 2-cell stage, more than 90% of the eggs was EGFP positive under fluorescent microscope. The injected 2-cell eggs was transferred into the oviduct of pseudo-pregnant female mice, and 42.9% (12/28) of the transferred mice became pregnant. The pregnant recipient mice gave birth to 120 pups, of which 60.8% (73/120) showed visible EGFP expression under UV light. The overall transgenic mouse production efficiency (transgenic mouse/injected eggs) was 5.0% (73/1 453). The EGFP transgenic founder mice were mated with wild-type mice, and EGFP expression was detected in F1, F2 and F3 offspring, indicating that the EGFP expression can be transmitted through germline cells.     Conclusion    Lentiviral transgenesis is an efficient and reliable method to produce transgenic mice. We have established the technological system for lentiviral transgenesis in mice.

参考文献/References:

刘勤,张晋宇,王露露,等. 慢病毒载体介导的增强型绿色荧光蛋白转基因小鼠的建立[J].第三军医大学学报,2008,30(10):885-889.

相似文献/References:

[1]潘万龙,方岩,许舸,等.结构特异性核酸酶FEN1对HBV复制的影响[J].第三军医大学学报,2012,34(19):1925.
 Pan Wanlong,Fang Yan,Xu Ge,et al.Effect of structure-specific nucleic acid enzyme FEN1 on HBV replication[J].J Third Mil Med Univ,2012,34(10):1925.
[2]赵亮,李静,魏强,等.人PGI基因siRNA慢病毒质粒的构建及对白血病细胞增殖的影响[J].第三军医大学学报,2013,35(01):20.
 Zhao Liang,Li Jing,Wei Qiang,et al.Construction of a lentiviral vector expressing human phosphoglucose isomerase gene siRNA and its influence on leukemia cell proliferation[J].J Third Mil Med Univ,2013,35(10):20.
[3]钱卫,彭代智,王丽华,等.慢病毒载体在人角质形成细胞基因组中的整合位点分析[J].第三军医大学学报,2013,35(01):24.
 Qian Wei,Peng Daizhi,Wang Lihua,et al.Analysis of lentiviral vector integration site preference in human keratinocytes[J].J Third Mil Med Univ,2013,35(10):24.
[4]秦勇,蔡金华,郑鹤琳,等.磁共振报告基因magA的慢病毒载体质粒构建及体外表达[J].第三军医大学学报,2011,33(13):1346.
 Qin Yong,Cai Jinhua,Zheng Helin,et al.Construction of recombinant lentivirus expression vector carrying MagA and its in vitro expression[J].J Third Mil Med Univ,2011,33(10):1346.
[5]郑钧丰,孔永,葛良鹏,等.2种腺相关病毒介导的增强型绿色荧光蛋白对人成纤维细胞转染效率的研究[J].第三军医大学学报,2008,30(12):1111.
 ZHENG Jun-feng,KONG Yong,GE Liang-peng,et al.AAV2-mediated versus AAV2/1-mediated enhanced green fluorescent protein transfection into human skin fibroblast in vitro[J].J Third Mil Med Univ,2008,30(10):1111.
[6]雒喜忠,王阁,郑继军,等.RAI16基因真核表达载体的构建及在HepG2中的表达[J].第三军医大学学报,2009,31(17):1620.
 LUO Xi-zhong,WANG Ge,ZHENG Ji-jun,et al.Construction of eukaryotic expression vector for RAI16 and its expression in HepG2 cells[J].J Third Mil Med Univ,2009,31(10):1620.
[7]林恒,唐春,吴乔,等.线粒体转录因子A对梗阻性黄疸大鼠肝功能的保护作用[J].第三军医大学学报,2009,31(22):2220.
 LIN Heng,TANG Chun,WU Qiao,et al.Protective effects of mitochondrial transcription factor A on hepatic function in rats with obstructive jaundice[J].J Third Mil Med Univ,2009,31(10):2220.
[8]周菁,徐瑜,张明慧,等.瘤内注射慢病毒载体介导的Bmi1-shRNA对A549皮下移植瘤的抑制作用[J].第三军医大学学报,2010,32(20):2177.
 Zhou Jing,Xu Yu,Zhang Minghui,et al.Intratumoral injection of recombinant lentivirus-mediated Bmi1-shRNA inhibits growth of A549 xenografts in mice[J].J Third Mil Med Univ,2010,32(10):2177.
[9]钟扬,李靖,黄小兵,等.慢病毒载体介导的人肝癌HepG2细胞BC047440基因沉默[J].第三军医大学学报,2010,32(08):744.
 Zhong Yang,Li Jing,Huang Xiaobing,et al.BC047440 silencing in HepG2 cells mediated by lentiviral vector[J].J Third Mil Med Univ,2010,32(10):744.
[10]罗静,蒋文,刘林,等.RPS14基因重组慢病毒载体的构建及其在MUTZ-8细胞中的表达[J].第三军医大学学报,2011,33(21):2252.
 Luo Jing,Jiang Wen,Liu Lin,et al.Construction of RPS14 recombinant lentiviral vector and its expression in MUTZ-8 cells[J].J Third Mil Med Univ,2011,33(10):2252.

更新日期/Last Update: 2008-05-26