Optimization of reporter gene assay:several factors influencing detection of promoter activity  

作　　者：XUE Li-xiang WENG Mo ZHANG Zong-yu TONG Tan-jun 

机构地区：[1]Department or Biochemistry and Molecular Biology,Health Science Center, Peking University, Beijing 100083, China

出　　处：《Chinese Medical Journal》2007年第11期965-969,共5页中华医学杂志（英文版）

基　　金：the grants from the National Basic Research Program of China (No. 2007CB507400);the National Natural Science Foundation of China (No. 30500082 and No.30671064)

摘　　要：Background Promoter analysis is currently applied to detect the expression of the targeted gene in studies of signal transduction and transcriptional regulation. As a reporter gene, luciferase plays an important role and has been used widely in the promoter assay. Methods Human embryonic lung fibroblast cells （2BS）, HeLa cells and MCF-7 cells were transfected with various genes embedded by lipofectamine. This study determined vadous factors that affect promoter activity determination, such as the selection of the reporter genes and internal references, the dose and the type of the vectors carrying the transcription factors, the host cells and the instruments. Results The sensitivity of the luciferase assay was much higher than that of enhanced green fluorescence protein （EGFP）. Moreover, promoter activity is increased in a dose-related manner only in certain ranges outside of which the results may be reversed and the promoter activity is related to the expression vector which is carrying the cDNA. Otherwise, the length of the promoter, internal references and the host cell can also influence the promoter activity. Conclusions To detect the promoter activity accurately, a few factors including dose, vector, length and host cell which influence reporter gene assay aforementioned should be considered.Background Promoter analysis is currently applied to detect the expression of the targeted gene in studies of signal transduction and transcriptional regulation. As a reporter gene, luciferase plays an important role and has been used widely in the promoter assay. Methods Human embryonic lung fibroblast cells （2BS）, HeLa cells and MCF-7 cells were transfected with various genes embedded by lipofectamine. This study determined vadous factors that affect promoter activity determination, such as the selection of the reporter genes and internal references, the dose and the type of the vectors carrying the transcription factors, the host cells and the instruments. Results The sensitivity of the luciferase assay was much higher than that of enhanced green fluorescence protein （EGFP）. Moreover, promoter activity is increased in a dose-related manner only in certain ranges outside of which the results may be reversed and the promoter activity is related to the expression vector which is carrying the cDNA. Otherwise, the length of the promoter, internal references and the host cell can also influence the promoter activity. Conclusions To detect the promoter activity accurately, a few factors including dose, vector, length and host cell which influence reporter gene assay aforementioned should be considered.

关 键 词：promoter regions activity assay LUCIFERASES green fluorescent proteins biological factors 

分 类 号：Q78[生物学—分子生物学]