title: AP-4F, antennapedia peptide linked to an amphipathic alpha helical peptide, increases the efficiency of Lipofectamine-mediated gene transfection in endothelial cells.
abstract: Typically, endothelial cells are difficult to transfect. In this study, we report that antennapedia peptide (AP) linked to L-4F, a water-soluble, amphipathic alpha helical peptide that avidly binds lipids (AP-4F) increases Lipofectamine 2000-mediated transfection of bovine coronary endothelial cell cultures. Transfection efficiency was monitored by flow cytometry and fluorescent microscopy. Lipofectamine 2000 transfection of endothelial cell cultures with green fluorescence protein (GFP)-DNA typically yields transfection efficiencies of 35.4+/-3.3% with low levels of cell death (8.1+/-1.0%). Pre-treatment of the Lipofectamine 2000-GFP-DNA complexes with AP-4F for 5 min increased transfection to 58.2+/-2.8% without increasing cell death. AP-4F increases Lipofectamine 2000-mediated transfection in a time-dependent fashion (within 10-20 min). Systematic studies reveal that the individual components of AP-4F, i.e., AP and L-4F alone, are ineffective in increasing Lipofectamine 2000-mediated transfection and that AP-4F must be directly associated with DNA liposomes prior to transfection for optimal uptake by endothelial cells. These observations demonstrate that AP-4F may be useful for increasing the transfection efficiency of endothelial cell cultures with standard commercially available reagents.
authors.affiliation: Division of Pediatric Surgery, Department of Surgery, Medical College of Wisconsin, Cardiovascular Center M4060, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
authors.forename: Jingsong
authors.initials: J
authors.lastname: Ou
authors.forename: Tracy
authors.initials: T
authors.lastname: Geiger
authors.forename: Zhijun
authors.initials: Z
authors.lastname: Ou
authors.forename: Allan W
authors.initials: AW
authors.lastname: Ackerman
authors.forename: Keith T
authors.initials: KT
authors.lastname: Oldham
authors.forename: Kirkwood A
authors.initials: KA
authors.lastname: Pritchard
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D051943:Antennapedia Homeodomain Protein; D002411:Cation Exchange Resins; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D018398:Homeodomain Proteins / Q000737:chemistry; D008055:Lipids; D008081:Liposomes; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors; D014162:Transfection / Q000379:methods*
chemical_list: C475156:AP-4F peptide; D051943:Antennapedia Homeodomain Protein; D002411:Cation Exchange Resins; D018398:Homeodomain Proteins; C472885:L-4F peptide; D008055:Lipids; C086724:Lipofectamine; D008081:Liposomes; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D010455:Peptides; D014157:Transcription Factors; D049452:Green Fluorescent Proteins