Replication-deficient adenovirus vector transfer of gfp reporter gene into supraoptic nucleus and subfornical organ neurons.
The present studies used defined cells of the subfornical organ (SFO) and supraoptic nuclei (SON) as model systems to demonstrate the efficacy of replication-deficient adenovirus (Ad) encoding green fluorescent protein (GFP) for gene transfer. The studies investigated the effects of both direct transfection of the SON and indirect transfection (i.e., via retrograde transport) of SFO neurons. The SON of rats were injected with Ad (2 x 10(6) pfu) and sacrificed 1-7 days later for cell culture of the SON and of the SFO. In the SON, GFP fluorescence was visualized in both neuronal and nonneuronal cells while only neurons in the SFO expressed GFP. Successful in vitro transfection of cultured cells from the SON and SFO was also achieved with Ad (2 x 10(6) to 2 x 10(8) pfu). The expression of GFP in in vitro transfected cells was higher in nonneuronal (approximately 28% in SON and SFO) than neuronal (approximately 4% in SON and 10% in SFO) cells. The expression of GFP was time and viral concentration related. No apparent alterations in cellular morphology of transfected cells were detected and electrophysiological characterization of transfected cells was similar between GFP-expressing and nonexpressing neurons. We conclude that (1) GFP is an effective marker for gene transfer in living SON and SFO cells, (2) Ad infects both neuronal and nonneuronal cells, (3) Ad is taken up by axonal projections from the SON and retrogradely transported to the SFO where it is expressed at detectable levels, and (4) Ad does not adversely affect neuronal viability. These results demonstrate the feasibility of using adenoviral vectors to deliver genes to the SFO-SON axis.
Vasquez
E C
EC
Department of Psychology, Cardiovascular Center, University of Iowa, Iowa City, Iowa, 52242, USA.
Johnson
R F
RF
Beltz
T G
TG
Haskell
R E
RE
Davidson
B L
BL
Johnson
A K
AK
D000256:Adenoviridae; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D051668:Delayed Rectifier Potassium Channels; D005260:Female; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000032:analysis / Q000378:metabolism*; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics*; D008297:Male; D008564:Membrane Potentials / Q000502:physiology; D009474:Neurons / Q000737:chemistry / Q000166:cytology / Q000502:physiology; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000502:physiology; D024642:Potassium Channels, Voltage-Gated; D051381:Rats; D017207:Rats, Sprague-Dawley; D013356:Subfornical Organ / Q000166:cytology*; D013495:Supraoptic Nucleus / Q000166:cytology*; D014162:Transfection; D014779:Virus Replication
D051668:Delayed Rectifier Potassium Channels; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D049452:Green Fluorescent Proteins