Hello,

We are using the specially optimized kit from amaxa for mouse BMDMs (VPA-1009) to transfect our gene of interest.

With the pGFP control plasmid supplied with the kit (3,5kb) we get about 30% of transfected macrophages, but with our plasmid of interest, we have 0% transfection, although the viability is really good (90%)

Our plasmid is 7,5kb long and contains our gene of interest under a CMV promoter, as well as a GFP/blasticidin resistance fusion gene under a human EF-1 promoter. After nucleofection, we detect absolutely no GFP expression.

Could this be due to the EF-1 promoter? In theory it should be active in mouse cells. Has anyone had any success with nucleofection of 7kb+ vectors in BMDMs?

Thank you!