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.