Endothelial cell DNA transfer and expression using Petri dish electroporation and the nonreplicating vaccinia virus/T7 RNA polymerase hybrid system
| Autor: | E W Lewis, A W Heinze, T J Rudo, MA Rahman St John, Bruce H. Howard, J L Chu, K A Engleka |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
Chloramphenicol O-Acetyltransferase
Umbilical Veins Green Fluorescent Proteins Immunoblotting Population Gene Expression Vaccinia virus Biology law.invention Viral Proteins law Bacteriophage T7 Gene expression Genetics medicine Humans T7 RNA polymerase education Molecular Biology education.field_of_study Electroporation Petri dish Genetic transfer Gene Transfer Techniques Receptors Interleukin-2 DNA-Directed RNA Polymerases Transfection Cell sorting Molecular biology Luminescent Proteins Microscopy Fluorescence Molecular Medicine Endothelium Vascular medicine.drug |
| Zdroj: | Gene Therapy. 6:1617-1625 |
| ISSN: | 1476-5462 0969-7128 |
| DOI: | 10.1038/sj.gt.3300977 |
| Popis: | 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 microg/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 (IL2Ralpha) pro- tein followed by anti-IL2Ralpha-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 IL2Ralpha-sorted population after transfection of T7 promoter-directed bicistronic IL2Ralpha/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. |
| Databáze: | OpenAIRE |
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