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 (10-40 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.
Lewis
E
E
Department of Physiology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107-6799, USA.
Rudo
T
T
St John
M R
MR
Chu
J
J
Heinze
A
A
Howard
B
B
Engleka
K
K
D017123:Bacteriophage T7; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases; D018274:Electroporation; D004730:Endothelium, Vascular / Q000378:metabolism*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D015375:Receptors, Interleukin-2 / Q000235:genetics; D014471:Umbilical Veins; D014616:Vaccinia virus / Q000235:genetics*; D014764:Viral Proteins
D008164:Luminescent Proteins; D015375:Receptors, Interleukin-2; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D015500:Chloramphenicol O-Acetyltransferase; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases