Optimization of reporter gene assay: several factors influencing detection of promoter activity.
Optimization of reporter gene assay: several factors influencing detection of promoter activity.
复制标题
报告基因测定的优化：影响启动子活性检测的几个因素。
DOI：
10.1097/00029330-200706010-00005
复制
发表时间：
2007
影响因子：
6.1
通讯作者：
T. Tong
中科院分区：
文献类型：
--
作者：
Li;Mo Weng;Zong;T. Tong
背景
目前，启动子分析在信号转导和转录调控研究中用于检测目标基因的表达。荧光素酶作为报告基因发挥着重要作用，在启动子检测中得到广泛应用。
方法
人胚胎肺成纤维细胞（2BS）、HeLa 细胞和 MCF-7 细胞用 lipofectamine 嵌入的各种基因转染。本研究确定了影响启动子活性测定的各种因素，如报告基因和内参的选择、携带转录因子的载体的剂量和类型、宿主细胞和仪器。
结果
荧光素酶检测的灵敏度远高于增强型绿色荧光蛋白（EGFP）。此外，启动子活性仅在某些范围内以剂量相关的方式增加，在该范围之外结果可能相反，并且启动子活性与携带cDNA的表达载体有关。另外，启动子的长度、内参和宿主细胞也会影响启动子活性。
结论
为了准确检测启动子活性，应考虑影响上述报告基因检测的一些因素，包括剂量、载体、长度和宿主细胞。
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.