摘要
目的:对比小鼠白蛋白(mouse albumin promoter,ALB)启动子调控下的增强型绿色荧光蛋白(en-hanced green fluorescent protein,EGFP)在不同细胞系中的转录活性。方法:以小鼠全血基因组DNA为模板,聚合酶链反应(polymerase chain reaction,PCR)扩增ALB启动子序列,克隆至pEGFP-1中,构建重组体pALB-EGFP;在Lipofectamine介导下将pALB-EGFP、pEGFP-N1转染人胎肝细胞L02、人宫颈癌细胞HeLa、人结肠癌细胞SW480、人胰腺癌细胞Bxpc-3;荧光显微镜和流式细胞仪对各转染细胞中EGFP的表达进行检测。结果:pALB-EGFP构建成功;L02转染pALB-EGFP72h后,ALB启动子可起始EGFP的表达,转录活性为人巨细胞病毒(cytomegalovir-us,CMV)启动子的1/4,其它转染细胞的ALB不能起始EGFP的转录;稳定筛选后,ALB的转录活性达到与CMV相当的水平。结论:构建的重组载体在肝脏来源细胞中具有较高的转录活性,为建立肝脏特异性表达目的基因的转基因小鼠模型奠定了基础。
AIM ： To study the tissue - specific function of albumin （ ALB ） in different cell lines. METH- ODS ： Total DNA was extracted from mouse blood for PCR amplification of ALB promoter. The amplified fragments were inserted into the multiple cloning sites of pMD - 18T vector and identified using restriction enzyme digestion and sequencing, then cloned into the expression vector pEGFP - 1. pALB - EGFP and its control pEGFP - N1 was transfected into the cell lines of L02, HeLa, SW480 and Bxpc - 3 by Lipofectamine 2000, respectively. The expressions of EGFP were detected using fluorescence microscopy and quantitatively with flow cytometry. RESULTS： ALB promoter was successfully cloned into the expression vector pEGFP - 1. ALB promoter drove EGFP expression in transfected L02 cells and product of EGFP was observed at 72 h after transfection. However, no expression of EGFP in the cell lines of HeLa, SW480 and Bxpc3 was observed after transfection with pALB - EGFP. The control expression vector pEGFP - N1, which contains the promoter of CMV, drove the expression of EGFP very well in all these 4 cell lines after transfection. Level of EGFP transient expression driven by ALB was only a quarter of that driven by CMV promoter in L02. However, after G418 selection, the level of EG- FP stable expression driven by ALB reached a considerable level as compared to that of CMV promoter. CONCLUSION ： Cloned mouse ALB promoter presents a strong transcriptional activity in L02 cells among the tested cell lines. It will serve as a suitable tool for transgenic mouse in the future.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2009年第7期1365-1369,共5页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.30471983)
