title: Effective plasmid DNA and small interfering RNA delivery to diseased human brain microvascular endothelial cells.
abstract: Expression of exogenous DNA or small interfering RNA (siRNA) in vitro is significantly affected by the particular delivery system utilized. In this study, we evaluated the transfection efficiency of plasmid DNA and siRNA into human brain microvascular endothelial cells (HBMEC) and meningioma cells, which constitute the blood-cerebrospinal fluid barrier, a target of meningitis-causing pathogens. Chemical transfection methods and various lipofection reagents including Lipofectamin™, FuGene™, or jetPRIME®, as well as physical transfection methods and electroporation techniques were applied. To monitor the transfection efficiencies, HBMEC and meningioma cells were transfected with the reporter plasmid pTagGFP2-actin vector, and efficiency of transfection was estimated by fluorescence microscopy and flow cytometry. We established protocols based on electroporation using Cell Line Nucleofector® Kit V with the Amaxa® Nucleofector® II system from Lonza and the Neon® Transfection system from Invitrogen resulting in up to 41 and 82% green fluorescent protein-positive HBMEC, respectively. Optimal transfection solutions, pulse programs and length were evaluated. We furthermore demonstrated that lipofection is an efficient method to transfect meningioma cells with a transfection efficiency of about 81%. Finally, we applied the successful electroporation protocols to deliver synthetic siRNA to HBMEC and analyzed the role of the actin-binding protein cortactin in Neisseria meningitidis pathogenesis.
authors.affiliation: Institute of Hygiene and Microbiology, University of Würzburg, Würzburg, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Slanina
authors.forename: M
authors.initials: M
authors.lastname: Schmutzler
authors.forename: M
authors.initials: M
authors.lastname: Christodoulides
authors.forename: K S
authors.initials: KS
authors.lastname: Kim
authors.forename: A
authors.initials: A
authors.lastname: Schubert-Unkmeir
mesh_terms: D001422:Bacterial Adhesion; D001921:Brain / Q000378:metabolism* / Q000473:pathology; D048430:Cell Shape; D002470:Cell Survival; D051356:Cortactin / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000008:administration & dosage* / Q000235:genetics; D018274:Electroporation / Q000379:methods; D042783:Endothelial Cells / Q000378:metabolism / Q000473:pathology*; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008055:Lipids / Q000737:chemistry; D008579:Meningioma / Q000378:metabolism / Q000473:pathology; D010957:Plasmids / Q000008:administration & dosage* / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000235:genetics; D014162:Transfection / Q000379:methods*
chemical_list: D051356:Cortactin; D008055:Lipids; C086724:Lipofectamine; D034741:RNA, Small Interfering; D004247:DNA