I have some +/+ and -/- MEFs of a certain gene and want to add back in WT and various mutant cDNA of this gene, to determine functionality. I was planning to transfect the various cDNA's/plasmids via Lipofectamine 2000. Do you think this is feasible? Does anyone know how easily, or difficult, it is to transiently transfect MEFs? Thank you!!!
We used viruses and the MEFs transfected straight away. It may be good to remember that some people may question your results if you rely solely on transient transfections. Good luck with the Lipofectamine 2000.
Mirsada,
I will be doing retrovirus first. I just have a construct not in a retroviral vector. I was planning to try and transfect and select with it's selectable marker. Thank you!
I have found limited success with lipid based transfection reagents (no more than 15% transfected cells with the TransIT 2020 reagent). I have had very robust expression using Amaxa nucleofection (closer to 50% transfected cells). Nucleofection requires some optimization though. There are two MEF transfection solutions supplied by Lonza and various possible transfection settings. Higher transfection efficiency and protein expression comes at the expense of increased cell death. You would have to find the protocol that works best for your system.
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