A dual color fluorescent reporter system for the real time detection of promoter activity
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Understanding the mechanisms controlling transcription of a gene requires the identification and characterization of its cis-acting regulatory elements. A highly useful approach to the identification and characterization of cis-acting elements has been the systematic coupling of genomic fragments to reporter constructs, so called “promoter bashing”. The expression from such reporters must be normalized for differences in transient transfection efficiency between cells and replicates. A novel dual color fluorescent reporter system to assay the promoter activity of a genomic DNA fragment of interest was established by cloning a Discosoma red fluorescent protein gene and a green fluorescent protein gene into a single vector, giving a system in which the ratio between red and green fluorescence is proportional to promoter activity. This system allows real time quantitative monitoring of promoter activity. We validated this approach by assaying the cis-acting regulatory potential of the peroxisome proliferators-activated receptor gamma2 gene.
KeywordsDiscosoma red protein Dual color fluorescent reporter system Green fluorescent protein Peroxisome proliferators-activated receptor gamma2 gene Promoter activity
This study was supported by the National “863” Program of China (Grant No. 2008AA101010), National Natural Science Foundation of China (Grant No. 30972080), National Key Technology R&D Program (Grant No. 2008ADB2B03-19), Keystone Project of Transfer gene in China (Grant No. 2009ZX08009-157B, 2008ZX08007-002, and 2009ZX08007-005B-07), Program of National Beef Cattle Industrial Technology System (Grant No. CARS-38), Basic and Foreland Technology Study Program of Henan Province (No. 072300430160).