Enrichment of transiently transfected mesangial cells by cell sorting after cotransfection with GFP

Early passage mesangial cells, like many other nonimmortalized cultured cel ls, can be difficult to transfect. We devised a simple method to improve th e efficiency of transient protein expression under the transcriptional cont rol of promoters in conventional plasmid vectors in rat mesangial cells. We used a vector encoding modified green fluorescent protein (GFP) and steril e fluorescence-activated cell sorting (FACS) to select a population consist ing of >90% GFP-expressing cells from passaged nonimmortalized cultures tra nsfected at much lower efficiency Only 10% transfection efficiency was note d with a beta-galactosidase expression vector alone, but cotransfection wit h GFP followed by FACS and replating of GFP(+) cells yielded greater than f ivefold enrichment of cells with detectable beta-galactosidase activity. To demonstrate the expression of a properly oriented and processed membrane p rotein, we cotransfected GFP with a natriuretic peptide clearance receptor (NPR-C) expression vector. Plasmid-dependent cell surface NPR-C density was enhanced by 89% after FACS, though expression remained lower in selected m esangial cells than in the CHO cell line transfected with the same vector. We conclude that cotransfection of rat mesangial cells with GFP, followed b y FACS, results in improvement in transient transfection efficiencies to le vels that should suffice for many applications.