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Endothelial cell DNA transfer and expression using Petri dish electroporation and the nonreplicating vaccinia virus/T7 RNA polymerase hybrid system

Gene Therapy volume 6, pages 1617–1625 (1999)Cite this article

Abstract

The nonreplicating vaccinia virus MVA/T7 RNA polymerase hybrid system was tested with Petri dish electroporation for ectopic gene expression in human umbilical vein endothelial cells (HUVECs). A range of voltages (150–450 V), pulse times (10–40 ms), DNA concentrations (0–20 μg/ml) and infection levels (0–15 multiplicities of infection) were tested for effects on T7 promoter-directed chloramphenicol acetyltransferase (CAT) activity after MVA/T7RP infection. MVA/T7RP-directed expression was transient and at least 10 000-fold in excess of nonviral, cytomegalovirus enhancer-directed expression. Use of a Petri dish electrode with the MVA/T7RP system showed increased viability compared with a cuvette electrode. Overexpression of interleukin-2 alpha subunit (IL2Rα) pro- tein followed by anti-IL2Rα-directed magnetic immunoaffinity cell sorting allowed isolation of the transfected population. The high fidelity of cellular sorting was shown by segregation of CAT activity in the IL2Rα-sorted population after transfection of T7 promoter-directed bicistronic IL2Rα/CAT DNA. Expression of a panel of proteins including the fluorophore green fluorescent protein as detected by fluorescence microscopy and p21cip1, p27kip1, pp60c-src, FGF-1, pRb, p107 and pRb2/p130 proteins was also achieved. Thus, use of the nonreplicating vaccinia virus/T7 RNA polymerase expression system with Petri dish electroporation is feasible for certain applications for the manipulation of HUVECs by gene transfer.

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Acknowledgements

This work was supported by a National Research Council Research Associateship to Kurt A Engleka while a postdoctoral fellow at the National Institutes of Health, institutional support from Thomas Jefferson University and by a grant from the American Heart Association. The authors would like to thank Donald J Fujita, University of Calgary, Vasily V Ogryzko, National Institutes of Health, and Antonio Giordano, Thomas Jefferson University for supplying plasmids, Linda S Wyatt and Bernard Moss, National Institutes of Health, for MVA/T7RP recombinant virus, Marilyn Woolkalis, Thomas Jefferson University for HUVECs and P Macke Consigny, Thomas Jefferson University for the use of a fluorescence microscope and imaging system and rabbit microvascular endothelial cells.

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Authors and Affiliations

  1. Department of Physiology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA, USA

    E W Lewis, T J Rudo, J L Chu, A W Heinze & K A Engleka

  2. Laboratory of Molecular Growth Regulation National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA

    MA Rahman St John & B H Howard

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Lewis, E., Rudo, T., St John, M. et al. Endothelial cell DNA transfer and expression using Petri dish electroporation and the nonreplicating vaccinia virus/T7 RNA polymerase hybrid system. Gene Ther 6, 1617–1625 (1999). https://doi.org/10.1038/sj.gt.3300977

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