肝脏特异性表达人遗传印记基因PEG10转基因载体的构建和鉴定  

The construction and identification of transgenic vector with human imprinted gene PEG10 specially expressed in mouse liver

作　　者：刘瑶[1] 林菊生[1] 熊杰[2] 谭锦泉[2] 张强[1] 常莹[1] 任精华[3] 

机构地区：[1]华中科技大学同济医学院附属同济医院肝病研究所,湖北武汉430030 [2]武汉大学基础医学院免疫学系 [3]华中科技大学同济医学院附属协和医院肿瘤中心

出　　处：《胃肠病学和肝病学杂志》2008年第12期982-986,共5页Chinese Journal of Gastroenterology and Hepatology

摘　　要：目的构建小鼠肝脏特异性表达人遗传印记基因PEG10的转基因载体pALB-PEG10-EGFP,为制备转基因小鼠做准备。方法RT-PCR扩增PEG10基因cDNA序列,克隆入T载体进行酶切、测序鉴定,后将其定向克隆至真核表达载体pALB-EGFP中ALB的下游,构建转基因载体pALB-PEG10-EGFP;在Lipofectamine介导下转染L02细胞,经G418抗性筛选,挑选阳性克隆并扩大培养;采用RT-PCR、Western blot、免疫细胞化学等方法分析PEG10在L02细胞中的表达和细胞内定位。结果酶切和测序结果表明pALB-PEG10-EGFP构建成功;稳定转染后的L02表达有PEG10的mRNA及蛋白,且主要定位于胞浆。结论重组体pALB-PEG10-EGFP的构建初步奠定了转基因小鼠制备的基础。Objective To construct human imprinted gene PEG10 by mouse liver-specific expression vector pALBEGFP, which is for the preparation of transgenic mouse. Methods Full length cDNA of PEG10 open reading frame （ORF） 1 was amplified and cloned into pMD-18T simple vector for sequencing. The recombinant ORF 1 was then sub- cloned into pALB-EGFP, to construct the transgenic vector pALB-PEG10-EGFP. Recombinant plasmid was stably transfected into L02 ceils via lipofectamine2000 and selected by G418. The expression levels of PEG10 mRNA and protein were identified by RT-PCR, Western blot and immunocytochemistry, respectively. Results Enzyme analysis and sequencing analysis confirmed that the target gene was in right position of pALB-EGFP. RT-PCR and Western blot showed that PEG10 mRNA and protein were expressed in stable transfected L02 cells. The subcellular localization of PEG10 was in the whole cytoplasm of stable transfected L02 cells. Conclusion The eukaryotic express vector containing human PEG10 gene was successfully constructed and expressed. It will serve as a suitable tool for transgenic mouse in the future.

关 键 词：遗传印记基因 PEG10 白蛋白启动子 转基因载体 鉴定 

分 类 号：R735.7[医药卫生—肿瘤]