Replication-deficient adenovirus vector transfer of gfp reporter gene intosupraoptic nucleus and subfornical organ neurons

The present studies used defined cells of the subfornical organ (SFO) and s upraoptic nuclei (SON) as model systems to demonstrate the efficacy of repl ication-deficient adenovirus (Ad) encoding green fluorescent protein (GFP) for gene transfer. The studies investigated the effects of both direct tran sfection of the SON and indirect transfection (i.e., via retrograde transpo rt) of SFO neurons. The SON of rats were injected with Ad (2 x 10(6) pfu) a nd 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 tran sfection 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 neu ronal (approximately 4% in SON and 10% in SFO) cells. The expression of GFP was time and viral concentration related. No apparent alterations in cellu lar morphology of transfected cells were detected and electrophysiological characterization of transfected cells was similar between GFP-expressing an d nonexpressing neurons. We conclude that (1) GFP is an effective marker fo r gene transfer in living SON and SFO cells, (2) Ad infects both neuronal a nd nonneuronal cells, (3) Ad is taken up by axonal projections from the SON and retrogradely transported to the SFO where it is expressed at detectabl e levels, and (4) Ad does not adversely affect neuronal viability. These re sults demonstrate the feasibility of using adenoviral vectors to deliver ge nes to the SFO-SON axis. (C) 1998 Academic Press.