小鼠Egr-1基因启动子的克隆及其辐射诱导基因表达  

Cloning the promoter of mouse Egr 1 gene and its radiation inducibility of gene expression

作　　者：魏道严[1] 陈诗书[1] 王中和[2] 

机构地区：[1]上海第二医科大学生物化学教研室,人类基因治疗研究中心上海200025 [2]上海第二医科大学附属第九人民医院,上海200011

出　　处：《中国癌症杂志》2000年第4期326-329,共4页China Oncology

基　　金：国家自然科学基金! ( 3970 4 4 0 )

摘　　要：研究克隆小鼠Egr 1基因启动子及其辐射诱导基因表达特性。方法 :用合成的一对引物 ,以BALB c小鼠基因组为模板 ,扩增出 44 9bp大小含 6个CC(A T) 6 GG基序的序列 ,通过亚克隆技术分别将其插入pBluescriptⅡ测序载体和pHGFP S6 5T报告载体中。利用测序载体对克隆的DNA序列进行分析 ,报告质粒载体经Li pofecTAMINE脂质体介导转染COS 7细胞后给予γ射线照射或H2 O2 处理 ,用流式细胞仪观察GFP表达阳性细胞数。结果 :PCR扩增的DNA片段经测序证实与文献报道完全一致 ,报告质粒转细胞实验结果提示 ,照射或H2 O2 处理可经Egr 1基因启动子而诱导GFP(绿色荧光蛋白 )的表达。结论 :成功地克隆了Egr 1基因启动子 ,经照射能诱导报告基因GFP的表达 。Purpose:To clone the promoter of Egr 1 gene of mouse and characterize its radiation inducibility of gene expresson.Methods:The Egr 1 promoter sequence containing six CC(A/T) 6GG motifs was amplified from genomic DNA of BALB/c mouse with PRC method, then it was subcloned in to sequencing vector of pBluescriptⅡ and reporter vector of phGFP S65T. The constructs were used respectively for DNA sequencing or transfecting COS 7 cells. After exposure to different doses of γ irradiation or concentrations of H 2O 2, the percentage of GFP expression positive cells in the transfected COS 7 cells was counted by flow cytometer. Results:DNA sequence analysis showed that nucleotide sequence of the cloned promoter fragment was identical to that reported previously. γ irradiation or H 2O 2treatment can induce GFP expression in COS 7 cells transfected with reporter vector. Conclusions:The promoter of Egr 1 gene with the characteristics of radiation inducibility of gene expression form mouse has been successfully cloned, thus paving a sound foundation for further research.

关 键 词：启动子 EGR-1基因 辐射诱导 肿瘤 基因治疗 

分 类 号：R730.51[医药卫生—肿瘤]