What is the reason behind choosing the reporter gene when experimenting on your gene of interest?

Question

I noticed within example experiments in class that different reporter genes are chosen to be inserted near your gene of interest to prove whether or not the gene is being expressed. For example, you may insert the gene for fluorescence next to your gene of interest so you know if it is transcribed or not by whether the organism's cells are fluorescent and to what degree they are fluorescing at.

I have noticed in some experiments that have multiple versions that in one case they use the fluorescent gene and in the next a different gene (for example lactose). Both portions of the experiment use almost the exact same steps so why would they not choose the same reporter gene?

Answer 1

Different genes will serve different purposes. For example, if you want to perform colocalization studies, then fluorescent genes like eGFP and DS-Red (or any variation of those, namely Emerald, mCherry, etc) will be quite useful, since you can use different filters on your microscope for the various fluorophores. For morphological assessments, perhaps a LacZ gene or alkaline phosphatase may be better suited, since the detection of these enzymes is compatible with standard histological techniques and you get usually much better contrast than with fluorescence. For quantification purposes, you would use probably firefly luciferase or CAT, since there are very good quantitative assays for these enzymes.

The decision will also depend on the degree of experience of the lab with each detection technique and even in the availability of the constructs. If a reporter is already made by the lab next door and if it fits your needs, you'll just use it, instead of creating your own, even if the reporter gene would not have been your first choice.

Answer 2

Reporter genes are also used when the experiment is trying to distinguish the effect of that gene that the scientist introduces (exogenous) and the same gene that the cell already has (endogenous). Ideally, one would like to choose a cell system that does not contain endogenous genes of interest because of this confounding effect, but it is not always feasible.

Reporter genes are also mainly used when it is impossible or very difficult to directly measure a gene of interest, especially if the gene of relatively obscure in the scientific literature. For example, cells that do not express a thymidine kinase gene are impossible to visualize by Positron Emission Tomography. It's not because there is anything wrong with the gene of interest, just that the cells need a way to retain a radioactive agent that can be detected by the PET machine. Likewise, fluorescent genes are very often used because there is often a need to see where the gene product is located within the cell, and the only way to do that is to combine it with something visible by microscopy which lets you see fluorescence and the cell structures. Lastly, a reporter gene is often used when the gene of interest is hard to detect because of limitations of commercial antibodies or detection kits, and the reporter, being very commonly used, is easy to detect with commercial products and antibodies.

Answer 3

Reporter genes have become an invaluable tool in studies of gene expression.A gene consists of two functional parts: One is a DNA-sequence that gives the information about the protein that is produced (coding region). The other part is a specific DNA-sequence linked to the coding region; it regulates the transcription of the gene (promoter). The promoter is either activating or suppressing the expression of the gene. The purpose of the reporter gene assay is to measure the regulatory potential of an unknown DNA-sequence.So it is quite helpful,it;s kind of reverse engeneering the function and then let know what exactly it does.