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

Am J Physiol. 1999 May;276(5):F777-85. doi: 10.1152/ajprenal.1999.276.5.F777.

Abstract

Early passage mesangial cells, like many other nonimmortalized cultured cells, can be difficult to transfect. We devised a simple method to improve the efficiency of transient protein expression under the transcriptional control of promoters in conventional plasmid vectors in rat mesangial cells. We used a vector encoding modified green fluorescent protein (GFP) and sterile fluorescence-activated cell sorting (FACS) to select a population consisting of >90% GFP-expressing cells from passaged nonimmortalized cultures transfected at much lower efficiency. Only 10% transfection efficiency was noted with a beta-galactosidase expression vector alone, but cotransfection with GFP followed by FACS and replating of GFP+ cells yielded greater than fivefold enrichment of cells with detectable beta-galactosidase activity. To demonstrate the expression of a properly oriented and processed membrane protein, 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 mesangial cells than in the CHO cell line transfected with the same vector. We conclude that cotransfection of rat mesangial cells with GFP, followed by FACS, results in improvement in transient transfection efficiencies to levels that should suffice for many applications.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenoviridae*
  • Animals
  • Atrial Natriuretic Factor / metabolism
  • Atrial Natriuretic Factor / pharmacology
  • Cell Separation / methods*
  • DNA, Complementary
  • Flow Cytometry
  • Gene Transfer Techniques*
  • Genes, Reporter
  • Glomerular Mesangium / cytology*
  • Green Fluorescent Proteins
  • Indicators and Reagents / metabolism*
  • Iodine Radioisotopes
  • Liposomes / genetics
  • Luminescent Proteins / genetics*
  • Peptide Fragments / pharmacology
  • Rats
  • Rats, Sprague-Dawley
  • Transfection
  • beta-Galactosidase / genetics

Substances

  • DNA, Complementary
  • Indicators and Reagents
  • Iodine Radioisotopes
  • Liposomes
  • Luminescent Proteins
  • Peptide Fragments
  • atrial natriuretic factor (4-23)NH2, de-Gln(18)-de-Ser(19)-de-Gly(20,22)-de-Leu(21)-
  • Green Fluorescent Proteins
  • Atrial Natriuretic Factor
  • beta-Galactosidase