pGLOW and LacZ Trouble

[BPLabscience]

Over the past few months our lab has been attempting to measure expression differences between promoter sequences through transfection. We have a pGLOW plasmid to measure GFP expression on our two sample groups and a pCMV-LacZ control to measure transfection efficiency and number of plasmids per sample. The trouble we have run into is that GFP is expressed only if our LacZ plasmid is not present. We think something about the LacZ trasnfection interferes with pGLOWs transfection, and only LacZ gets transfected into the cells. I have a couple of questions:

 

First: Has anyone come across this problem before? If so, how did you manage to fix it?

 

Second: I find that pGLOW is rarely cited in publications as the experimental plasmid, has anyone had trouble with using pGLOW for dual transfection? Is there a better plasmid system to use to measure the effect of a SNP on gene expression?

 

Any help would be appreciated.

[CharonY]

Have you validated the presence of both vectors in your cell lines?

[BPLabscience]

We have only validated LacZ. Is there a way of validating GFP without measuring excitation/emission?

 

 

Have you validated the presence of both vectors in your cell lines?

[bioGOD]

We have only validated LacZ. Is there a way of validating GFP without measuring excitation/emission?

 

 

 

 

Correct me if I'm wrong, but doesn't pGLO contain an amp gene as the selection marker?

[BPLabscience]

Yes. And we select for that when growing up our bacterial colonies. We are also using pGLOW, not pGLO

[CharonY]

The simplest way would probably a PCR assay. pCMV and pGlow both have Amp resistances, I think. I assume you are testing it in an eukaryotic cell line? It is probably less of an issue, but IIRC pGlow is a pUC derivative that belongs to the same compatibility group as the pCMV vectors (both derived from pMB1). So they would not replicate in the same cell. But again, that is not an issue when measuring transfection into eukaryotic cells. However, in most methods co-transfection occurs at a lower rate and the smaller vector can outcompete the larger ones.

Edited December 6, 2011 by CharonY