Gene transfection of primary mouse hepatocytes in 384-well plates

We report the development of an improved in vitro transfection assay to test the efficiency of non-viral vector DNA nanoparticle transfection of primary hepatocytes. The protocol describes the isolation of viable hepatocytes from a mouse by collagenous perfusion. Primary mouse hepatocytes are plated in 384-well plates and cultured for 24 h prior to transfection with polyethylenimine (PEI) or peptide DNA nanoparticles. Luciferase expression is measured after 24 h following the addition of ONE-Glo substrate. The gene transfer assay for primary hepatocytes was optimized for cell plating number, DNA dose, and PEI to DNA ratio. The assay was applied to compare the expression mediated by mRNA relative to two plasmids possessing different promoters. The reported assay provides reliable in vitro expression results that allow direct comparison of the efficiency of different non-viral gene delivery vectors.