Recent Developments of Reporter Gene Assays - Methods and Protocols (with Appilications)

Reporter Genes are genes whose products can be readily assayed consecutive to transfection and can be used as markers for screening successfully transfected cells, for studying regulation of gene expression, or serve as controls for standardizing transfection efficiencies.

Gene reporter systems play a key role in gene expression and regulation studies. This review describes the ideal reporter systems, including reporter expression vector design. It summarizes the many uses of genetic reporters and outlines the currently available and commonly used reporter systems. Each system is described in terms of the reporter gene, the protein it encodes, and the assays available for detecting presence of the reporter. In addition, each reporter system is analyzed in terms of its recommended uses, advantages, and limitations.

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A gene consists of two main functional elements: The first consists of a so-called coding DNA sequence that provides information about the protein produced. Second, a specific promoter sequence linked to the coding region that regulates the transcription of the gene. The promoter serves to activate or suppress the expression of the gene as required.

The main purpose of the reporter gene assay is to investigate the promoter of a gene of interest, i.e. the regulation of its expression. This can be done by linking the promoter of interest to an easily detectable gene, such as the gene for firefly luciferase, which catalyses a reaction that produces light. 

Usually, the cells are then exposed to different factors or conditions, or changes can be made in the order of the reporter, the effect of which can be easily tracked by measuring changes in light emission.

EXAMPLES OF REPORTER GENES

Common reporter genes are β-galactosidase, β-glucuronidase and luciferase. Various detection methods (see below) are used to measure expressed reporter gene protein. These include luminescence, absorbance and fluorescence.

Assays based on luminescence are very popular for several reasons:

• they have a high sensitivity (between 10 and 10,000 times higher than methods based on absorption or fluorescence, depending on the specific assay and reporter used)
• most cell types do not have endogenous luciferase activity
• luminescence assays have a large dynamic range
• they are quick to perform
• their costs are relatively low 

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