16.11: Reporter Genes

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.

Commonly used reporter genes are - GFP (green fluorescent protein gene), lacZ (β- galactosidase gene), RFP (red fluorescent protein gene), and Luc (luciferase gene). The cells expressing GFP glow green when excited by wavelengths of blue to the ultraviolet range, while those expressing RFP glow red when excited by wavelengths of 488 nm or 532 nm. The cells expressing the Luc gene produce luciferase enzymes that catalyze a reaction with luciferin to produce light. The lacZ gene is the most common reporter gene used in E.coli. It produces an enzyme β-galactosidase that causes the bacteria to appear blue when grown in a media containing X-gal substrate.

Reporter genes to study promoter activity

Besides the use of reporter genes in studying gene expression and localization, they can also help in studying the strength of promoters. This is done by placing a reporter gene downstream of the promoter in a plasmid and then introducing it into the mammalian cell. The expression of the reporter gene monitors the activity of the promoter. If the reporter gene expression is high, the promoter is said to be a strong promoter; it’s weak if the reporter gene expression is low.

Reporter gene, when placed downstream of the promoter in a plasmid, also helps in measuring transfection efficiency. The recombinant plasmid is transfected into a mammalian cell, and its expression shows that the cell is successfully transfected. The amount of protein produced by the recombinant plasmid shows its transfection efficiency.

Researchers use reporter genes to determine when and where a gene of interest is expressed. A reporter gene codes for a protein that can be tracked, such as a protein with a known enzymatic activity or one that is fluorescent.

Gene expression is controlled by cis-regulatory sequences located upstream or downstream of a gene’s coding region in DNA .

The pattern and timing of gene expression can be determined by creating recombinant DNA with a reporter gene under the control of a cis-regulatory sequence of interest and introducing it into cells or an organism.

This recombinant gene is introduced into different cell types, and both the gene of interest and the reporter gene are allowed to express.

Since the gene of interest  and the reporter gene have the same cis-regulatory sequence, they are expressed in the same cells and at the same time. So, by monitoring the expression of the reporter gene, a scientist can track where the gene of interest is being expressed.

One of the well-known reporter genes is one that encodes for a protein called green fluorescent protein - or GFP.

GFP was discovered in the jellyfish, Aequorea victoria. The protein produces a green fluorescence under ultraviolet light, which enables researchers to track its location within a cell.

For example, to study the expression of β-tubulin in C. elegans, the coding sequence of the β-tubulin gene was replaced by the gfp gene.

The gfp gene expression is now under the control of the promoter for the β-tubulin gene.

This recombinant DNA was introduced into the worm using a microinjection, and the gene was expressed, similarly to β-tubulin.

The location of GFP was monitored using fluorescence microscopy. As GFP fluorescence was shown in the touch receptor neurons, this is likely a site where β-tubulin is expressed.