多顺反子慢病毒载体介导编码基因在人胚胎干细胞中的表达  

Multicistronic Lentiviral Vector Mediated High Expression of Encoded Genes in Human Embryo Stem Cell

作　　者：李慧[1] 李威[1] 王倩[2] 宋和存[1] 郭毅[1] 

机构地区：[1]中国医科大学附属一院妇科,中国辽宁沈阳110001 [2]中国医科大学附属一院检验科,中国辽宁沈阳110001

出　　处：《生命科学研究》2009年第2期133-136,共4页Life Science Research

基　　金：教育部留学归国人员科研课题;辽宁省教育厅科研课题资助项目(2004F036)

摘　　要：人胚胎干细胞的基因修饰对于干细胞在体外的调控及促进干细胞在治疗方面的应用具有很高价值.探讨了以艾滋病病毒-1为基础的慢病毒载体转导人胚胎干细胞,表达2种或3种转移基因的可行性和功效.应用脑心肌炎病毒的内部核糖体进入位点(IRES)序列和/或口蹄疫病毒(FMDV)裂解因子2A构建双顺反子和三顺反子慢病毒载体,绿色荧光蛋白(eGFP)和O6-甲基鸟嘌呤-DNA-甲基转移酶作为检测基因,抗嘌呤基因(嘌呤-N-甲基转移酶;PAC)作为选择基因,转染人胚胎干细胞后经过嘌呤选择.通过流式细胞技术和蛋白印迹杂交方法检测eGFP和MGMT的表达.多基因慢病毒载体可以同时将2或3个基因转移到人胚胎干细胞.多顺反子慢病毒载体转导人胚胎干细胞后经过嘌呤选择,可以使转移基因高效、均匀的表达,在人胚胎干细胞的基础和应用研究方面具有重要意义.Genetic modification of human embryonic stem cell （hESCs） is highly valuable not only for manipulating the cells in vitro but also promoting a therapeutic application. Here the feasibility and efficiency of expressing two or three transgenes by HIV-1 based lentiviral vector （LV） in human embryonic stem cells were explored. Bicistronic and tricistronic LVs were constructed employing the internal ribosomal entry site （IRES） sequence of encephalomyocarditis virus （EMCV） and/or foot-and-mouth disease virus （FMDV） cleavage factor 2A. Enhanced green fluorescent protein （eGFP） and O^6-methylguanine-DNA- methyhransferase were chosen （MGMT） as reporter genes and puromycin resistance gene（puromycin-Nacetyhransferase; PAC） as selectable gene to transduce hESCs then selected by puromycin. The expression of eGFP and MGMT by means of flow cytometry and Western blot were checked. The muhigene LVs were able to simultaneously transfer two or three genes into hESCs. Multicistronic LVs enable high and homogeneous expression of transgenes by polyclonal hESCs after puromycin selection. These vectors may provide important roles in basic and applied research on hESCs.

关 键 词：人胚胎干细胞 慢病毒载体 内部核糖体进入位点 裂解因子2A 甲基转移酶 

分 类 号：R318.04[医药卫生—生物医学工程]