Gene reporters enable valuable insight into gene expression. The GUS gene reporter system is one of the popular and common plant reporter systems. GUS, is short for glucuronidase, an enzyme in the bacterium E. coli. GUS is a good reporter for plants, as it does not occur naturally, and thus, has a low background. With some simple genetic techniques, one can attach the promoter of the gene you want to investigate to the GUS coding region. You can then transform your reporter construct into your plant species of choice to monitor its expression. Transformation can be accomplished in plants via methods like Agrobaterium-mediated gene transfer.
More Than One Assay for GUS Gene Reporter System
Good ol’ GUS is a consumer of carbohydrates, breaking down complex sugars into smaller fragments. Since we know this, we can provide the carbohydrates in media and predict how the GUS will be processing them. The two most common carbohydrate assay methods in use are the X-Gluc and MUG assays. How do you know which assay to utilize? That depends on your research question!
X-Gluc for Localization of Stain
If you are more concerned about the physical location where your gene expresses, I highly recommend X-Gluc. X-Gluc (chemists know it as 5-Bromo-4-chloro-3-indolyl-?-D-glucuronide) is a carbohydrate substrate for GUS, that when cleaved produces an intense blue colored precipitate. This is easily observable.
For example, in my Arabidopsis picture below, you can see the trichomes are a brilliant blue color while the epidermal leaf tissue is not. This tells us that the promoter gene driving our reporter GUS is expressed in trichomes but not in epidermis or vein tissues in the leaf.
X-gluc is great for visual identifications of where the gene expresses, as well as, if the gene is active or not. While X-Gluc makes very pretty pictures, the assay takes a while to complete due to the need to de-stain the tissue completely (24 – 48 hrs). It is also difficult to quantify, so if you want to qualitatively measure expression location use X-Gluc. But if you are wanting to quantitatively measure expression of your gene of interest, you should use MUG.
MUG for Quantitation of Gene Expression
MUG (4-Methylumbelliferyl ?-D-Galactopyranoside) is also a carbohydrate, but instead of producing a precipitate when it is cleaved by GUS, it produces a compound that fluoresces, aka glows. Using a spectrofluorometer, this glow is measurable. By adding the same amount of total protein and MUG substrate to the assay, the one variable between your samples is the amount of GUS. This last one is controlled by your gene of interest. The only way fluorescence occurs is by GUS enzyme activity. If you measure fluorescence over time you should get a linear curve. You want to express your data in terms of relative fluorescence released per minute per µg of protein.
Benefits of MUG assays are they are relatively quick (4 – 6 hrs) and quantitative. Downsides of a MUG assay is the cumbersome nature of data analysis due to the need to perform dozens of linear fits. This is why I like R, because I can use a simple code to get all of the data versus having to plot and linear fit each sample in Excel. When you have a 96 well plate that can get tedious. Another downside is this assay gives you no information on the physical location where the gene expresses, as the entire tissue sample is ground up into the assay. For example, the homogenization of leaf tissue into the assay makes it impossible to distinguish if the signal was coming from the veins, epidermis, trichome, etc.
I highly recommend utilizing the GUS gene reporter system: if you want to track down individual gene expression and can generate some transgenic plants. Use this system to identify the physiological location of gene expression via X-gluc assay or to quantify the expression levels by MUG analysis. This reporter is truly a one stop shop!
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