How does it take long your reporter gene?

The persistence of your reporter gene activity need to be seriously addressed when interpreting data within living cells. You should know the half life of your reporter (e.g., the time the host system need to inactivate half of reporter activity). Usually "short reporters" are preferred because, being rapidly inactivated, they are dynamically close monitored molecular mechanism; nevertheless sometimes monitoring a fragile-reporter is tricky, so a researcher that does not need to sharply know what happen in the cell every hour, can increase the sensitivity by choosing "long reporters" who accumulate into the cell  increasing sensitivity. Both academia and private companies are attempting to modify reporter's half life: good results have been obtained by eliminating (or inserting) degradation domains like PEST, and so obtaining stabilized (destabilized) reporter variants. Unfortunately, no reliable half lives tables can be drowned from the literature: the behavior of each reporter is dependent of the system, so it is important to know how to measure the half life in your host system. How to?






A common practice is to make a time-course study in which the activity of the reporter 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. 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 individual 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.