I've been facing problems in transfecting Mouse embrionic fibroblast (MEFs). I am using pcDNA 3.1 Hygro vector with (5600 bp) plus the insert (~3000 bp). As control I use pcDNA GFP to confirm the transfection efficiency. The transfection ratio (DNA: lipofectamine) are around 1:7 and 1:8. However, I've never got considerable transfection efficiency. As soon as I started the Hygromycin selection (two days after transfection) the cells die and I am using the lowest Hygromycin concentration (certified by antibiotic killing curve). I know that these cells hard to transfect cells but I've been trying this more than 6 months and with no sucess.
I would really appreciate any advice from those who has experience with this cell line.
Thank you very much in advance,