polyA before IRES - bad or OK? - (Feb/01/2006 )

Hi!

I've made a construct of the form: cDNA polyA IRES2-EGFP polyA
Essentially, I'm trying to express GFP and my gene of interest from the same plasmid so that any cell that gets transfected will express my gene and GFP.

I am not getting any expression of the GFP. Is the polyA before the IRES messing up transcription and/or translation of the GFP?

Or does the polyA have no effect on the expression of GFP?

Any help in troubleshooting this would be greatly appreciated!

Thanks!

not too sure about the mechanism, but the poly A holds a stop transcription capacity. So if you have a poly A transcription may ends few 10's base down it?...

i'm not sure that the DNA will be transcribed enough to reach the IRES...

Hi-

I currently use this system. I didn't include any poly A at the end of the gene in my first cistron (my gene of interest). I think Fred is right. polyAA before the IRES I think stops transcription. Besides the resulting mRNA from the vector will alredy have a poly A in it after EGFP(it's part of the vector).

I remember reading something similar to this before on this forum....uh hopefully this link works

http://www.protocol-online.org/forums/inde...ic=2571&hl=IRES




Good Luck,

Mountainman

Thanks for the link. It looks like I was re-inventing the wheel.

I'm going to try and re-engineer the construct to get rid of the polyA sequence before the IRES. I'll re-transfect and see if it works!
I'll keep everyone posted.

Thanks for all the feedback!

I don't want to create a fusion protein, just in case the GFP messes up the function of my gene.
Plus, then I'd also have to do extra controls to show the GFP fusion protein works the same as the protein itself.

I'm looking at expression on a cellular level, so doing a cotransfection won't necessarily work because some cells will get the GFP + my gene, and some will get GFP without my gene.

Thanks for the feedback!