Components of the eukaryotic vaccinia virus/T7 RNA polymerase hybrid expression system were assessed using recombinant and nonrecombinant forms of modified vaccinia Ankara (MVA), a replication-deficient vaccinia virus strain. Recombinant MVA virus expressing T7 RNA polymerase (Wyatt, L. S., Moss, B., and Rozenblatt, S. (1995). Virology 210, 202-205) stimulated high levels of expression from a T7 promoter-chloramphenicol acetyltransferase (CAT) reporter. Most, but not all, of the virally induced expression was T7 RNA polymerase and T7 promoter dependent, with no viral enhancement of translation of T7 transcripts. The efficacy of supplying T7 RNA polymerase expression from nonviral sources was evaluated using a self-amplifying T7 RNA polymerase autogene or an inducible T7 RNA polymerase expression vector. The latter modes yielded CAT activity dependent on T7 RNA polymerase expression; however, expression required viral factors independent of T7 RNA polymerase and did not reach that attained using the recombinant virus. In further experiments, MVA-induced T7 RNA polymerase expression was upregulated by alpha-amanitin, an inhibitor of eukaryotic polymerases. This indicates that MVA/T7 RNA polymerase hybrid expression may be rendered still more efficient by ameliorating transcriptional interference due to an alpha-amanitin-sensitive eukaryotic factor(s).
The nonreplicating vaccinia virus MVA/T7 RNA polytein followed by anti-IL2R␣-directed magnetic immunoafmerase hybrid system was tested with Petri dish electropfinity cell sorting allowed isolation of the transfected popuoration for ectopic gene expression in human umbilical vein lation. The high fidelity of cellular sorting was shown by endothelial cells (HUVECs). A range of voltages (150-segregation of CAT activity in the IL2R␣-sorted population 450 V), pulse times (10-40 ms), DNA concentrations (0-after transfection of T7 promoter-directed bicistronic 20 g/ml) and infection levels (0-15 multiplicities of IL2R␣/CAT DNA. Expression of a panel of proteins includinfection) were tested for effects on T7 promoter-directed ing the fluorophore green fluorescent protein as detected chloramphenicol acetyltransferase (CAT) activity after by fluorescence microscopy and p21 cip1 , p27 kip1 , pp60c-src , MVA/T7RP infection. MVA/T7RP-directed expression was FGF-1, pRb, p107 and pRb2/p130 proteins was also achitransient and at least 10 000-fold in excess of nonviral, eved. Thus, use of the nonreplicating vaccinia virus/T7 cytomegalovirus enhancer-directed expression. Use of a RNA polymerase expression system with Petri dish elecPetri dish electrode with the MVA/T7RP system showed troporation is feasible for certain applications for the increased viability compared with a cuvette electrode. manipulation of HUVECs by gene transfer. Overexpression of interleukin-2 alpha subunit (IL2R␣) pro-
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