Hi I don't think I have ever come across a single gene with two promoters in the vector before therefore not sure how much any comments I make would make sense. However is it possible that having two promoters in close proximity might mean that proteins binding one promoter might block the binding of the second promoter pomoter (if that makes sense)?
There is an interesting article in PLosOne comparing promoter strengths:http://www.plosone.org/article/info:doi ... ne.0010611
and the pgk promoter is fairly weak. Perhaps it would be safer (?) just to swap out the pgk for another promoter (e.g. CMV).
However saying that the paper above suggests that CMV was one of the most variable (cell type to cell type) and we have had a frustrating time with this promoter inactivating (almost certainly due to methylation) in H9C2 muscle precursor cells over time (this has been reported before - although we discovered the article after we had problems).
Another article (http://www.ncbi.nlm.nih.gov/pubmed/17609656
) suggests that some promoters may act in a cell-stage specific fashion so if transfecting stem cells maybe the two promoter system may work better (?).
Anyway let us know what the expression is like (if you do compare single promoters to two together it may make a nice little paper!). Good luck!