RNAi targeting one gene in a bicistronic transcript - Will the other gene be knock down? (Jan/29/2007 )
My RNAi target gene was cloned in a expression vector and was followed by IRES and gfp. When cotransfected with shRNA, will gfp be knocked down too? Thank you!
If it were a fusion protien, the whole mRNA transcript would be knock down.
But with IRES, i think the GFP will be transcribed independent of the preceding gene, so I don't think GFP will be knockdown.
I think you would knock down both:
IRES: An internal ribosome entry site, abbreviated IRES, is a nucleotide sequence that allows for translation initiation in the middle of a messenger RNA (mRNA) sequence as part of the greater process of protein synthesis.
Therefore the mRNA gets cleaved after siRNA recognition and so the whole mRNA is degraded. In this case no Ribosome can attach to the IRES because the mRNA is degraded and no GFP is produced.
Promega uses a similar system for sequence validation (psiCHECK2) where Luciferase is cloned in front of your gene (no ires there i have to admit) but there is a stop codon between them and if you target either Luc or your gene, the other one dissapears as well.
correct me if i'm wrong!
Thank for your reply.
I am going to test 5 shRNA against my target gene. After co-transfection, I don't see gfp level decrease. Though I think it should.
Anyway, I will do a western blot to comfirm.
I had poblems with this approach in the past... GFP is extremely stable in (t 1/2 over 24 h) the cell and it is really hard to see knockdown. In my case it only worked with a control sirna against GFP and in some stable cell lines. If you can use destabilized GFP (d2EGFP for example) otherwise it might not work. Or switch to Luciferase, SEAP, or any other marker. If you are trying to test your shRNAs in transient transfection, make sure you have a control to check transfection efficiency (if you only get 50 % of the cells you might not be able to see anything ever)