Poly A Signals and Fusion Proteins

[dgibson]

Hi everybody,

This is my first time posting here. I'll try to provide as much relevant info as possible without getting too detailed.

I have cloned a gene (let's call it GENE for the sake of clarity) into the MCS of the pEGFP-N1 vector from Clontech. The GENE is followed by a poly A signal, the whole of which is floxed, yielding: loxP-GENE-polyA-loxP-EGFP-polyA. The EGFP is followed by its own poly A in the vector.

If my thinking is correct, the first poly A (after GENE) should prevent the extension of the mRNA into the EGFP coding sequence and, thus, prevent the expression of EGFP in cells following transfection. After Cre-mediated recombination, the GENE-polyA will be excised and EGFP will be expressed. Is this correct so far?

After transfection into COS-7 cells without the presence of Cre, I see very strong EGFP expression when I should see none. In a different well of the same experiment I used a construct that contained a "Cre-polyA-IRES-mCherry" cassette, and the poly A signal in that case prevented the expression of mCherry because the IRES site was never transcribed.

These seem like conflicting results and I'm not sure what's going on. Any thoughts? I appreciate your help!