Optimization of reporter gene assay: several factors influencing detection of promoter activity.
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 various 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.
Xue
Li-xiang
LX
Department of Biochemistry and Molecular Biology, Health Science Center, Peking University, Beijing 100083, China. lixiangxue@bjmu.edu.cn
Weng
Mo
M
Zhang
Zong-yu
ZY
Tong
Tan-jun
TJ
D002478:Cells, Cultured; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D008156:Luciferases / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014162:Transfection
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008156:Luciferases