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

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 (10-40ms), DNA concentrations (0-20 mu g/ml) and infection levels (0-15 multiplicities of infection) were tested for effects on T7 promoter-directed chloramphenicol acetyltransferase (CAT7) 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-e alpha subunit (IL2R alpha) protein followed by anti-IL2R alpha-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 IL2R alpha-sorted population after transfection of T7 promoter-directed bicistronic IL2R alpha/CAT DNA. Expression of a panel of proteins including the fluorophore green fluorescent protein as defected by fluorescence microscopy and p21(cip1), p27(idp1), pp60(c-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.