Silencing of the first cistron - (Feb/26/2010 )
Our lab uses a bicistronic vector linking our gene of interest (cistron 1) to luciferase (cistron 2) with an ECMV-IRES. Expression is driven by CMV promoter.
We use luciferase expression through monitoring of bioluminescence both in vitro and in vivo as a surrogate for monitoring expression of cistron 1.
While our cells are still bioluminescent, it seems that cistron 1' expression has decreased dramatically.
My PI is now telling me that there can be silencing of the first cistron (at the transcriptional level) with the IRES still promoting luciferase transcription, hence the bioluminescence observed... I am afraid she may have misunderstood the person who told her that but I just wanted to double check in case I was missing something.
Any input or advices would be welcome!
Thanks a bunch!
IRES-dependent reporter gene expression is not stoichiometric to first gene expression (see for example PMID 10933956). It depends on both first and second gene themselves, I had higher expression of IRES-eGFP than of the first gene. If you want to quantify first gene expression to reporter gene expression, I'd recommend using a 2A system. Here's some reference: http://www.virologyj.com/content/3/1/14 (PMID 16539700).