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Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
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A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells

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Isotopic assays for reporter gene activity.

R E Kingston¹, J Sheen, D Moore

¹Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.

Current Protocols in Molecular Biology

|February 12, 2008

Summary

This study details two radioactive reporter gene assays for measuring gene expression and protein secretion. The chloramphenicol acetyltransferase (CAT) assay quantifies gene expression, while the human growth hormone (hGH) radioimmunoassay measures protein secretion.

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Area of Science:

Molecular Biology
Biochemistry
Cell Biology

Background:

Reporter gene assays are crucial for quantifying gene expression and protein secretion in cellular studies.
Radioactive detection methods offer high sensitivity for these measurements.
Established assays like chloramphenicol acetyltransferase (CAT) and human growth hormone (hGH) are widely utilized.

Purpose of the Study:

To describe two established radioactive reporter gene assays for assessing gene expression and protein secretion.
To provide detailed methodologies for the chloramphenicol acetyltransferase (CAT) assay and the human growth hormone (hGH) radioimmunoassay.

Main Methods:

Chloramphenicol acetyltransferase (CAT) assay: Measures CAT enzyme activity in cell lysates using radioactive chloramphenicol and acetyl CoA, with separation via thin-layer chromatography or phase-extraction.
Human growth hormone (hGH) radioimmunoassay: Quantifies secreted hGH in culture medium using a two-site assay with a radiolabeled antibody and avidin-coated beads for immune complex collection.

Main Results:

The CAT assay allows for the determination of transfected gene expression levels.
The hGH radioimmunoassay enables accurate quantification of secreted human growth hormone.
Both assays provide reliable and sensitive detection of biological molecules.

Conclusions:

Radioactive reporter assays, including CAT and hGH assays, are effective tools for studying gene expression and protein secretion.
These methods provide quantitative data essential for molecular biology and cell biology research.
The described protocols offer a foundation for researchers utilizing these sensitive detection systems.