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

Abstract

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-