Comparison of nonviral transfection and adeno-associated viral transduction on cardiomyocytes
Cardiomyocytes are terminally differentiated cells that to date have been characterized as poor targets for nonviral gene transfer. This study was therefore designed to determine the optimal nonviral gene transfer parameters in cell cultures of neonatal rat cardiomyocytes and to compare them with the efficiency of gene transfer using adeno-associated viral vectors (AAV). Transfection efficiency was measured by quantitative chloramphenicol acetyltransferase type I (CAT)-enzyme-linked immunosorbent assay and β-galactosidase staining, based on overexpression of reporter genes (CAT and LacZ).
The efficiency of CAT/LacZ overexpression was assessed using the following techniques: (1) liposomal reagents, such as: FuGENE 6, LipofectAMINE 2000, LipofectAMINE PLUS, GenePORTER, Metafectene, and LipoGen; (2) electroporation and nucleofector techniques; and (3) an AAV2 vector harboring a lacZ reporter gene. Toxicity was monitored by total protein measurement and by analyzing cell metabolism.
On average, Lipofectamine 2000 was the most effective nonviral method examined yielding consistently high transfection rates (8.1% β-galactosidase-positive cells) combined with low toxicity. Electroporation also resulted in high transfection values (7.5%); however, cellular toxicity was higher than that of Lipofectamine 2000. Finally, transduction with AAV2 vectors provided the highest levels of transduction (88.1%) with no cellular toxicity.
We conclude that although transduction with AAV is more efficient (88.1%), transfections with nonviral techniques, when optimized, may provide a useful alternative for overexpression of therapeutic genes in neonatal cardiomyocytes.
Index EntriesLiposomes lipofection electroporation gene therapy nucleofection adeno-associated virus
Felgner, P. L. (1991). Cationic liposome-mediated transfection with lipofectin reagent. In Gene Transfer and Expression Protocols
, vol. 7, Methods in Molecular Biology
(Murray, E. J., ed.), Humana Press, Clifton, NJ, pp. 81–89.Google Scholar
Blaese, R. M., Mullen, C. A., and Ramsey, W. J. (1993) Strategies for gene therapy. Pathol. Biol. (Paris)
, 672–676.Google Scholar
Albert, N. and Tremblay, J. P. (1992) Evaluation of various gene transfection methods into human myoblast clones. Transplant Proc.
, 2784–2786.PubMedGoogle Scholar
Dodds, E., Dunckley, M. G., Naujoks, K., Michaelis, U., and Dickson, G. (1998) Lipofection of cultured mouse muscle cells: a direct comparison of lipofectamine and DOSPER. Gene Ther.
, 542–551.PubMedCrossRefGoogle Scholar
Trivedi, R. A. and Dickson, G. (1995) Liposome-mediated gene transfer into normal and dystrophin-deficient mouse myoblasts. J. Neurochem.
, 2230–2238.PubMedCrossRefGoogle Scholar
Vitiello, L., Bockhold, K., Joshi P. B, and Worton, R. G. (1998) Transfection of cultured myoblasts in high serum concentration with DODAC:DOPE liposomes. Gene Ther.
, 1306–1313.PubMedCrossRefGoogle Scholar
Kott, M., Haberland, A., Zaitsev, S., Buchberger, B., Morano, I., and Bottger, M. (1998) A new efficient method for transfection of neonatal cardiomyocytes using histone H1 in combination with DOSPER liposomal transfection reagent. Somat. Cell Mol. Genet.
, 257–261.PubMedCrossRefGoogle Scholar
Aihara, H. and Miyazaki, J. (1998) Gene transfer into muscle by electroporation in vivo. Nat. Biotechnol.
, 867–870.PubMedCrossRefGoogle Scholar
Mir, L. M. (1999) High-efficiency gene transfer into skeletal muscle mediated by electric pulses. Proc. Natl. Acad. Sci. USA
, 262–267.CrossRefGoogle Scholar
Mathiesen, I. (1999) Electropermeabilization of skeletal muscle enhances gene transfer in vivo. Gene Ther.
, 508–514.PubMedCrossRefGoogle Scholar
Block T. A. (1995) Nonthermally mediated muscle injury and necrosis in electrical trauma. J. Burn Care Rehabil.
, 581–588.PubMedCrossRefGoogle Scholar
Lee, R. C., Canaday D. J., and Hammer, S. M. (1993) Transient and stable ionic permeabilization of isolated skeletal muscle cells after electrical shock. J. Burn Care Rehabil.
, 528–540.PubMedCrossRefGoogle Scholar
Green, N. K., Franklyn, J. A., Ohanian, V., Heagerty, A. M., and Gammage, M. D. (1997) Transfection of cardiac muscle: effects of overexpression of c-myc and c-fos proto-oncogene proteins in primary cultures of neonatal rat cardiac myocytes. Clin. Sci. (Lond.)
, 181–188.Google Scholar
Monahan, P. E. and Samulski, R. J. (2000) Adeno-associated virus vectors for gene therapy: more pros than cons? Mol. Med. Today
, 433–440.PubMedCrossRefGoogle Scholar
Wollert, K. C., Taga, T., Saito, M., et al. (1996) Cardiotrophin-1 activates a distinct form of cardiac muscle cell hypertrophy: assembly of sarcomeric units in series VIA gp130/leukemia inhibitory factor receptor-dependent pathways. J. Biol. Chem.
, 9535–9545.PubMedCrossRefGoogle Scholar
Davidson, B. L., Stein, C. S., Heth, J. A., et al. (2000) Recombinant adeno-associated virus type 2, 4, and 5 vectors: transduction of variant cell types and regions in the mammalian central nervous system. Proc. Natl. Acad. Sci. USA
, 3428–3432.PubMedCrossRefGoogle Scholar
Brisson, M. and Huang, L. (1999) Liposomes: conquering the nuclear barrier. Curr. Opin. Mol. Ther.
, 140–146.PubMedGoogle Scholar
Simoes, S., Pires, P., Duzgunes, N., and Pedrosa de Lima, M. C. (1999) Cationic liposomes as gene transfer vectors: barrier to successful application in gene therapy. Curr. Opin. Mol. Ther.
, 147–157.PubMedGoogle Scholar
Clackson, T. (2000) Regulated gene expression systems. Gene Ther.
, 120–125.PubMedCrossRefGoogle Scholar
Cameron, J., Iversen, N., Torsdalen, K., Birkenes, B., and Djurovic, S. (2003) Electroporation is the best nonviral transfection technique in human endothelial and smooth muscle cells
, submitted.Google Scholar
Bureau, M. F., Gehl, J., Deleuze, V., Mir, L. M., and Scherman, D. (2000) Importance of association between permeabilization and electrophoretic forces for intramuscular DNA electrotransfer. Biochim. Biophys. Acta
, 353–359.PubMedGoogle Scholar
Vicat, J. M. (2000) Muscle transfection by electroporation with high-voltage and short-pulse currents provides high-level and long-lasting gene expression. Hum. Gene Ther.
, 909–916.PubMedCrossRefGoogle Scholar
Hoover, F. and Kalhovde, M. J. (2000) A double-in-jection DNA electroporation protocol to enhance in vivo gene delivery in skeletal muscle. Anal. Biochem.
, 175–178.PubMedCrossRefGoogle Scholar
Somia, N. and Verma, I. M. (2000) Gene therapy: trials and tribulations. Nat. Rev. Genet.
, 91–99.PubMedCrossRefGoogle Scholar