Reporter Gene Assays
Reporter genes are genes whose products can be readily assayed subsequent 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.
The ideal reporter gene should be absent from the cells used in the study or easily distinguishable from the native form of the gene, assayed conveniently, and have a broad linear detection range. It is also important that the presence of the reporter gene does not affect the normal physiology and general health of the transfected cells.
Reporter genes can be expressed constitutively or inducably with an external intervention such as the introduction of IPTG in the β-galactosidase system. Generally, reporter gene assays are performed 1–3 days after transfection; however the optimal time for the assay should be determined empirically.
In contrast to selectable markers, which protect an organism from a selective agent that would normally kill it or prevent its growth, reporter genes used for screening transfectants make the cells containing the reporter gene visually identifiable. Reporter genes used in this way are normally expressed under their own promoter independent from that of the introduced gene of interest, allowing the screening of successfully transfected cells even when the gene of interest is only expressed under certain specific conditions or in tissues that are difficult to access.
Reporter genes can also serve as controls for transfection. For example, transfection efficiencies between different experiments can be normalized by comparing the expression levels of a reporter gene used in all of the experiments.
Gene regulation assays
Reporter gene assays are invaluable for studying regulation of gene expression, both by cis-acting factors (gene regulatory elements) and trans-acting factors (transcription factors or exogenous regulators). Furthermore, reporter gene systems enable the use of pathway-specific, tissue-specific, or developmentally regulated gene promoters as biomarkers for specific events processes.
In these assays, the detectable reporter gene acts as a surrogate for the coding region of the gene under study. The reporter gene construct contains one or more gene regulatory elements to be analyzed, the sequence for the reporter gene, and the sequences required for the transcription of functional mRNA. Upon introduction of the reporter construct into cells, expression levels of the reporter gene are monitored through a direct assay of
the reporter proteins enzymatic activity.
Common reporter genes
Commonly used reporter genes that induce visually identifiable characteristics usually involve fluorescent and luminescent proteins.
Green fluorescent protein (GFP)
Green fluorescent protein (GFP) causes cells that express it to glow green under UV light. A specialized microscope is required to see individual cells. Yellow and red versions are also available, allowing the investigation of multiple genes at once. It is commonly used to measure gene expression.
Luciferase as a laboratory reagent often refers to P. pyralisluciferase, although recombinant luciferases from several other species of fireflies are also commercially available. The luciferase enzyme catalyzes a reaction with its substrate (usually luciferin) to produce yellow-green or blue light, depending on the luciferase gene. Since light excitation is not needed for luciferase bioluminescence, there is minimal autofluorescence and thus virtually background-free fluorescence.
GUS assay (using β-glucuronidase) is an excellent method for detecting a single cell by staining it blue without using any complicated equipment. The drawback is that the cells are killed in the process. It is particularly common in plant science.
Blue-white screen is used in both bacteria and eukaryotic cells. The bacterial lacZ gene encodes a β-galactosidase enzyme. When media containing certain galactosides (e.g., X-gal) is added, cells expressing the gene convert the X-gal to a blue product and can be seen with the naked eye.