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- Title
Endothelial cell DNA transfer and expression using Petri dish electroporation and the nonreplicating vaccinia virus/T7 RNA polymerase hybrid system.
- Authors
Lewis, E W; Rudo, T J; St John, MA Rahman; Chu, J L; Heinze, A W; Howard, B H; Engleka, K A
- 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.
- Subjects
ENDOTHELIUM; ELECTROPORATION; VACCINIA; GENETIC transformation; GENE expression
- Publication
Gene Therapy, 1999, Vol 6, Issue 9, p1617
- ISSN
0969-7128
- Publication type
Article
- DOI
10.1038/sj.gt.3300977