ABC of reporter genes

Written in stream of consciousness, please have fun!

Alkaline phosphatase, Beta-galactosidase and Chloramphenicol-acetil-transferase are three ancient reporter genes that, although characterized by a narrow linear Dynamic range, and ampered by some host's Endogenous activity, have been widely used in molecular biology. Today, Fluorescent proteins, seem to catch the scenario of research literature, but new reporters have been introduced, like Glucanases. The Half-life of such reporters addresses their future use, while Interaction between two reporter could spread out new applications (look at resonance energy transfer). Up to date, to the best of my knowledge, no Journals are devoted to reporter gene developments, this could partially explain the need for a blog tracking research highlights. But this blog will track also other questions like where and how you Knock your transgene. My favourite reporter genes are Luciferases: although to date they don't have the high resolution of MRI techniques, they benefit of low signal-to-Noise ratio also when adopted in Optical imaging, and this have been demonstrated with more than one Promoter. Although some Quenching can happen in vivo, new Red-shifted luciferases have been described in order to minimize tissue absorption and increase Sensitivity. Another trend recorded in 2007 was the development of multimodality reporters, for instance one can exploit the same promoter to express both luciferase for optical imaging and Thymidine-kinase for positron-emission tomography. More than one reporter can be assessed with more than one technique, this means lot of Unit of measures both in Vitro and in Vivo, some authors use to track reporter gene expression with Western blot also in 2007 (oh my God!). At least one reporter gene is sexy (secreted XYlanase). Now, let me open a contest: find the best Z-word and suggest it as a comment, don't forget to mention your blog, I will review in a dedicated post the blog of the winner.

How does it takes long your reporter gene?

Persistence of your reporter gene activity need to be seriously addressed when interpreting data within living cells. To know the half life of your reporter is mandatory (the time the host system need to inactivate half of reporter activity). Usually "short reporters" are preferred because, being rapidly inactivated, they are dynamic and closest to report the expression machinery activity in real-time; nevertheless sometimes monitoring a fragile-reporter is tricky, so researcher that doesn't need sharply to know what happen in the cell every hour, can increase the sensitivity choosing "long reporters": those last ones, accumulating into the cell can overcame the threshold of sensitivity. A big body of attempts to tailor the half life of reporter genes have been performed both from academia and companies: generally good results have been obtained eliminating (or inserting) degradation domains like PEST obtaining stabilized (destabilized) reporter variants. Unfortunately, no reliable tables of half lives can be drowned from literature, because each reporter variant can be subjected to differential inactivation in different cells, so it is important to know how to measure half lives in your host system. How to?

The common practice is to make a time-course study in which the activity of the reporter (eventually previously induced) is monitored after the treatment with an inhibitor of protein synthesis (cycloheximide, CHX). Although average reporter degradation rates have been usually measured for population of cells, techniques that only measure population averages obscure the variation that exists between cells. This limitation will give you some pain when you will observe variability in reporter signal and no clues will be available to guess whether such variability is due to synthesis or degradation. Halter and colleagues from the National Institute of Standards and Technology (NIST) recently introduced in Citometry (DOI: 10.1002/cyto.a.20461) an automated microscopy on micropatterned arrays that confined cell migration and allows to segment the cells using phase contrast images. With such method, eGFP signal arising from ~500 single cells cultured in rings of 44 micrometer was monitored, enabling accurate measurement of degradation rates in individual cells and making it possible to determine the distribution of degradation rates within the clonal population. These measurements of GFP half-lives in statistically significant numbers of cells provide assurance that higher GFP abundance in some cells is due to higher rates of gene expression and not due to lower rates of protein degradation. Micropatterning techniques shows all their charme.