Abstract
Primary cells are a valuable tool for researchers and are often preferred over transformed or immortalized cell lines since they are biologically more relevant and resemble the in vivo situation much closer. Unfortunately, efficient gene transfer in primary cells is still limited. Whereas viral strategies are time consuming and involve safety risks, nonviral methods are often inefficient for most primary cells. Nucleofection has been proven to overcome these limitations. Here, we describe the Nucleofection protocol for efficient transfection of human umbilical vein endothelial cells. Using a combination of a cell type-specific solution and electrical conditions, transfection efficiencies up to 90% can be achieved while survival rate is more than 70%.
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References
Haleem-Smith, H., Derfoul, A., Okafor, C., et al. (2005) Optimization of high-efficiency transfection of adult human mesenchymal stem cells in vitro. Mol. Biotechnology 30, 9–20.
Kimmelman, J. (2005) Recent developments in gene transfer: risk and ethics. BMJ 330, 79–82.
Williams, D., and Baum, C. (2003) Gene Therapy – New Challenges Ahead. Science 302, 400.
Thomas, C. E., Ehrhardt, A., and Kay, M. A. (2003) Progress and problems with the use of viral vectors for gene therapy. Nat. Rev. Genet. 4(5), 346–58.
Lakshmipathy, U., Buckley, S., and Verfaillie, C. (2007) Gene transfer via nucleofection into adult and embryonic stem cells. Methods Mol. Biol. 407, 115–26.
Zeitelhofer, M., Vessey, J.P., Xie, Y., et al. (2007) High-efficiency transfection of mammalian neurons via nucleofection. Nat. Protoc. 2(7),1692–704.
Landi, A., Babiuk, L.A., and van Drunen Littel-van den Hurk S. (2007) High transfection efficiency, gene expression, and viability of monocyte-derived human dendritic cells after nonviral gene transfer. J Leukoc Biol. 82(4), 849–60.
Gerber, S.A., and Pober, J.S. (2008) IFN-alpha induces transcription of hypoxia-inducible factor-1alpha to inhibit proliferation of human endothelial cells. J. Immunol. 181(2), 1052–62.
Kapoor, M., Zhou, Q., Otero, F., et al. (2008) Evidence for annexin II/S100A10 complex and plasmin in mobilization of cytokine activity of human TrpRS. J. Biol. Chem. 283(4), 2070–2077.
Gresch, O., Engel, F.B., Nesic, D., et al. (2004) New non-viral method for gene transfer into primary cells. Methods 33, 151–163.
Browne, C.D., Hindmarsh, E.J., and Smith, J.W. (2006) Inhibition of endothelial cell Âproliferation and angiogenesis by orlistat, a fatty acid synthase inhibitor. FASEB J. 20(12), 2027–35.
Benny, O., Fainaru, O., Adini, A., et al. (2008) An orally delivered small-molecule formulation with antiangiogenic and antiÂcancer activity. Nat Biotechnol. 26(7), 799–807.
Chen, Y., Wei, T., Yan, L. (2008) Developing and applying a gene functional association Ânetwork for anti-angiogenic kinase inhibitor activity assessment in an angiogenesis co-culture model. BMC Genomics. 9, 264–279.
Fan, F., and Wood, K.V. (2007) Bioluminescent assays for high-throughput screening. Assay Drug Dev. Technol. 5, 127–136.
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Gresch, O., Altrogge, L. (2012). Transfection of Difficult-to-Transfect Primary Mammalian Cells. In: Hartley, J. (eds) Protein Expression in Mammalian Cells. Methods in Molecular Biology, vol 801. Humana Press. https://doi.org/10.1007/978-1-61779-352-3_5
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DOI: https://doi.org/10.1007/978-1-61779-352-3_5
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