Enhanced green fluorescent protein as an alternative control reporter to Renilla luciferase.
Enhanced green fluorescent protein as an alternative control reporter to Renilla luciferase.
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DOI：
10.1016/j.ab.2005.04.047
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发表时间：
2005-07
影响因子：
2.9
通讯作者：
F. Vesuna;P. Winnard;V. Raman
中科院分区：
文献类型：
--
作者：
F. Vesuna;P. Winnard;V. Raman
Reporter gene assays facilitate analysis of transcriptional activity [1]. In such experiments, the initial characterization of the transcriptional activity of the gene product of interest is analyzed through its ability to bind to its cognate promoter sequence and either activate or repress a reporter gene’s activity. This is achieved by cloning the target promoter sequence upstream of a reporter gene. This construct is referred to as the experimental reporter. Measurement of the activity of the experimental reporter (ie, assaying reporter gene product levels) in the presence of the gene of interest (effector gene) reflects effector gene product activity. Variations in the transcriptional activity of the effector gene are then reflected in changes in the levels of expression of the reporter gene. To quantitate the reporter gene activity accurately, the transfection efficiency of all plasmids must be taken into account. One of the most common methods of determining transfection efficiency is to introduce and measure the activity of a second reporter gene (control reporter) under the control of a constitutive promoter within the same experimental setting.Reporter genes that have been used to determine transfection efficiency include chloramphenicol acetyl transferase (CAT), 1 β-galactosidase, luciferase, and green fluorescent protein (GFP)[2–4]. The luciferase reporter gene system is widely used due to its detection sensitivity and the ease of quantification when compared with the other available systems [5]. The luciferase gene from the sea pansy Renilla reniformis under the control of either simian virus 40 (SV40), cytomegalovirus (CMV), or thymidine kinase (TK) promoter is routinely used for this purpose. The Renilla control reporter, when cotransfected into cells with the experimental reporter and the effector construct, allows the evaluation of experimental variation such as transfection efficiency and cell viability. In general, for these assay systems, luciferase from the firefly Photinus pyralis is used as the experimental reporter. The advantage of this dual luciferase system is that because the substrate requirements of the two luciferases are unique, their respective expression levels can be estimated sequentially from within the same sample.
