Expression problems with pIRES vector from Clontech - EGFP at MCSB site cannot be expressed! (Aug/25/2005 )

Hi, everyone:

I am currently using the pIRES vector from Clontech/BD bioscience (Cat no. 631605), and the vector struction is pCMV-MCSA-IRES-MCSB. I cloned the EGFP cDNA (salI and NotI fragment, from pEGFP1 vector, BD Biosciences) into the MCSB site of the pIRES vector. then I cloned other gene of interest into the MCSA sites. sequencing confirmed that the structure of the constructs are correct.

Then, the problem come up: There is no green fluorescence under fluorescent microscope after transfecting the plasmid into Hela cells!! (hela cells were routinely cultured in DMEM with 10% FBS plus 5mM sodium pyruvate and 100ug/ml penicillin and streptomycin)

Is it true that the pIRES vector series from Clontech/BD Biosciences are all craps???? I have swopped the EGFP from MCSB site to MCSA site of the pIRES vector, then at MCSA site, it works fine, with lots of green fluorescent cells after transfection. So, it seems that the MCSB site have a serious problems with expressing proteins! So, maybe the IRES from MCMV has something wrong when the staff in Clontech engineers the vector?

Does anyone get the same problem in expressing proteins from the MCSB site using the IRES vector series from Clontech/BD Biosciences? If it does work in your hands, could you please tell me which particular vector you used and how did you manage to make it work at the MCSB site?

In addition, since the IRES is from encephalomyocarditis virus (ECMV) as described by the datasheet from BD Biosciences, does this vector only work in some particular cell lines? It gave me expression that many people used COS, 293 cells for bi-cistronic plasmids transfection, and not many people use Hela cells?

thanks very much!!

Hello!
Did you manage to get the pIRES vector to work from Clontech?
Thanks
Julia