Meet AGATA:

Meet AGATA:

21.10.14, 17h00 Venture Challenge EPFL, Lausanne. Closed.
27.10.14, 17h00 Sictic Investor Day, Lausanne CH. Program.
28.10.14, 18h00 Bioscope inauguration, Geneva CH. Program.

How to improve reporter activity

ResearchBlogging.orgGot a weak promoter in exam and your reporter signal is very close to background? Welcome to the club. Discovery companies tried to approach this problem designing for you more stable reporters (that accumulate over time), but they ask you to sacrifice any dynamic curiosity about what is going to happen over time. So what you can do?
In 1988 some viral sequences were described to initiate ribosome binding and translation in a cap-independent manner, they were named IRES (Internal Ribosome Entry Site) and they opened the way for bicistronic vectors. So, lot of scientist make construct like this:
PROMOTER-GENE1-IRES-GENE2-polyA
One single promoter drive a double transcript containing two messengers; then the IRES is supposed to drive to the ribosome the GENE2 alone with an efficiency ranged between 20% and 50% of the cap-dependent GENE1. This constitute the major disadvantage of bicistronic reporter.

When Bouabe and colleagues from Munich University comes to the club of weak promoter, they wondered: "Why don't put another copy of the same reporter after the IRES?".
PROMOTER-REPORTER-IRES-REPORTER-polyA
Such approach is described in Nucleic Acid Research and constitutes a simple technology that allows improvement of reporter gene activity using multiple IRES-Reporter linked in tandem. Basically IRES allow the same weak promoter to express more reporter without interfering with its transcriptional dynamics. Fluorescent proteins like EGFP (that to date, need to be at least 10,000-100,000 molecules/cell in order to be visualized) are probably the reporter that would benefit more of such improvement, without the need to touch their stability.

---/ citation /--- --- ---
Bouabe, H., Fassler, R., & Heesemann, J. (2008). Improvement of reporter activity by IRES-mediated polycistronic reporter system Nucleic Acids Research, 36 (5) DOI: 10.1093/nar/gkm1119