Vaccinia virus infection and gene transduction in cultured neurons.
The study of neurons in culture would benefit from the development of a gene transduction system capable of delivering foreign genes at high efficiency, as transduction of primary neurons with existing systems is inefficient. The efficacy of lytic vaccinia virus (VV) infection of primary retinal cultures and PC12 cells (a model of neuronal differentiation) was examined in order to determine the efficiency of gene transduction using VV in neuronal primary culture. VV was able to infect retinal cells and PC12 cells and express transgenes of Escherichia coli beta-galactosidase (lacZ) and epithelial fatty acid binding protein (E-FABP) in a virus dose-dependent manner. Most (50-100%) of the retinal cells were positive for transgene protein at multiplicities of infection (MOI) between 10 and 100 plaque-forming units (PFU), while over 50% of VV-infected PC12 cells expressed the virus encoded gene at an MOI = 10. The production of foreign mRNA and protein by VV following infection was verified by PCR and Western blot. Because VV is a lytic virus, cytopathic effects were examined. Retinal cultures maintained for 0.5 days in vitro showed greater than 90% survival at 24 h post-infection, while 14-day cultures were equally viable for 48 h. Retinal ganglion cells and differentiated PC12 cells appear to be more protected against lytic VV infection than proliferating glial and undifferentiated PC12 cells. These data suggest that VV may be a useful vector for delivering foreign genes to neuronal cells with an efficient transient transgene expression.
Allen
Gregory A
GA
Department of Physiology, School of Medicine, Loma Linda University, Loma Linda, CA, USA.
Denes
Bela
B
Fodor
Istvan
I
De Leon
Marino
M
D000818:Animals; D015153:Blotting, Western; D002470:Cell Survival; D002478:Cells, Cultured; D029968:Escherichia coli Proteins / Q000032:analysis / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D009457:Neuroglia / Q000737:chemistry / Q000821:virology*; D009474:Neurons / Q000821:virology*; D016716:PC12 Cells; D012329:RNA, Bacterial / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D012165:Retinal Ganglion Cells / Q000737:chemistry / Q000821:virology*; D014161:Transduction, Genetic / Q000379:methods*; D014616:Vaccinia virus / Q000235:genetics* / Q000502:physiology*; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D001616:beta-Galactosidase