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Effective plasmid DNA and small interfering RNA delivery to diseased human brain microvascular endothelial cells

Effective plasmid DNA and small interfering RNA delivery to diseased human brain microvascular endothelial cells
Effective plasmid DNA and small interfering RNA delivery to diseased human brain microvascular endothelial cells
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.
1464-1801
245-257
Slanina, H.
695cab74-446a-44b3-887f-7d93368e3317
Schmutzler, M.
f797a573-70b6-4e4a-a464-d24a7b556bb8
Christodoulides, M.
eba99148-620c-452a-a334-c1a52ba94078
Kim, K.S.
b292b4f8-c2c6-4719-bfc9-47a3a2138801
Schubert-Unkmeir, A.
4e0a589d-5038-4a59-8a1a-ffe3cff0cd30
Slanina, H.
695cab74-446a-44b3-887f-7d93368e3317
Schmutzler, M.
f797a573-70b6-4e4a-a464-d24a7b556bb8
Christodoulides, M.
eba99148-620c-452a-a334-c1a52ba94078
Kim, K.S.
b292b4f8-c2c6-4719-bfc9-47a3a2138801
Schubert-Unkmeir, A.
4e0a589d-5038-4a59-8a1a-ffe3cff0cd30

Slanina, H., Schmutzler, M., Christodoulides, M., Kim, K.S. and Schubert-Unkmeir, A. (2012) Effective plasmid DNA and small interfering RNA delivery to diseased human brain microvascular endothelial cells. Journal of Molecular Microbiology and Biotechnology, 22 (4), 245-257. (doi:10.1159/000342909). (PMID:23036990)

Record type: Article

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.

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Published date: 2012
Organisations: Clinical & Experimental Sciences

Identifiers

Local EPrints ID: 344306
URI: http://eprints.soton.ac.uk/id/eprint/344306
ISSN: 1464-1801
PURE UUID: 6286c739-0ebc-4702-8445-4a6275d570b2
ORCID for M. Christodoulides: ORCID iD orcid.org/0000-0002-9663-4731

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Date deposited: 17 Oct 2012 13:55
Last modified: 15 Mar 2024 02:39

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Contributors

Author: H. Slanina
Author: M. Schmutzler
Author: K.S. Kim
Author: A. Schubert-Unkmeir

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