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为研究基因枪对体外培养的哺乳动物肿瘤细胞系和经小鼠皮肤转移基因效率及转基因的表达量和表达时间,探讨作为一种新的基因转移工具,基因枪在基因治疗中应用的可能性.其方法:①用Helios基因枪分别对体外培养的4种人肿瘤细胞系:肺腺癌GLC-82、结肠癌HCF、膀胱癌EJ、肝癌QSG-7701和1种小鼠黑色素瘤细胞系B16,转携带报告基因Lac-Z的质粒pAdEldCMV-LacZ和携带报告基因ECFP(绿色荧光蛋白突变体)的质粒pEGFPC1,分别用x-gal染色法(Lac-Z)和直接荧光显微镜观察法检测基因转导效率和报告基因的表达.②用Helios基因枪直接对C57BL/6小鼠腹部皮肤转pAdE1dCMV-LacZ,3d后,断颈法处死小鼠,取转基因部位皮肤做冰冻切片,x-gal染色观察转基因的表达深度和表达量.③用Helios基因枪直接对C57BL /6小鼠腹部皮肤转携带鼠GM-CSF基因的质粒pWRG3142,转基因后6h,1,2,3,4,
To investigate the efficiency of gene guns in mammalian tumor cell lines cultured in vitro and in mouse skin and the expression and expression time of transgenes, to explore the possibility of using gene guns in gene therapy as a new gene transfer tool. Methods: (1) Four human tumor cell lines cultured in vitro using Helios gene gun: lung adenocarcinoma GLC-82, colon cancer HCF, bladder cancer EJ, liver cancer QSG-7701, and mouse melanoma cell line B16 The plasmid pAdEldCMV-LacZ carrying the reporter gene Lac-Z and the plasmid pEGFPC1 carrying the reporter gene ECFP (green fluorescent protein mutant) were used to detect gene transfer by x-gal staining (Lac-Z) and direct fluorescent microscopy, respectively. Expression efficiency and reporter gene expression.2 Hematos gene gun was used to directly transfer pAdE1dCMV-LacZ to the abdominal skin of C57BL/6 mice. After 3 days, the mice were sacrificed by cervical dislocation. The skin of the transgene site was frozen and observed by x-gal staining. Expression and depth of transgene expression.3 Helicobacter gene gun was used to directly transfer plasmid pWRG3142 carrying mouse GM-CSF gene to abdomen skin of C57BL/6 mice, 6h after transgene, 1, 2, 3, 4