Adenovirus-mediated gene transfer to cultured nodose sensory neurons

Abstract

Recent advances have enabled transfer of genes to various types of cells and tissues. The goals of the present study were to transfer genes to nodose sensory neurons using replication-deficient adenovirus vectors and to define the conditions needed to optimize the gene transfer. Neurons were dissociated from rat nodose ganglia and maintained in culture. Cultures were exposed for 30 min to vectors containing the β-galactosidase gene lacZ driven by either the Rous sarcoma virus (RSV) or the cytomegalovirus (CMV) promoter. Cultures were fixed and treated with X-gal to evaluate lacZ expression 1–7 days after exposure to virus. Increasing concentrations of virus led to dose-related increases in the number of neurons expressing lacZ. LacZ was expressed in 8±2, 39±6, and 82±3% of neurons 1 day after exposure to 10 7, 10 8, and 10 9 pfu/ml of AdRSV lacZ, respectively ( P<0.05). The same doses of AdCMV lacZ led to expression in 41±9, 60±10, and 86±4% of neurons. Expression driven by the CMV promoter was essentially maximal within 1 day and remained stable for at least 7 days. In contrast, expression driven by the RSV promoter was less on day 1 but increased over time (1–7 days). There was no lacZ expression in vehicle-treated cultures and exposure to the adenovirus vectors did not adversely influence cell viability. Exposure of the neuronal cultures to an adenovirus vector containing the gene for green fluorescent protein (AdRSV gfp, 10 9 pfu/ml) enabled visualization of successful gene transfer in living neurons. The results indicate that gene transfer to cultured nodose neurons can be accomplished using adenovirus vectors. The expression of the transferred gene persists for at least 7 days, occurs more rapidly when expression is driven by the CMV compared with the RSV promoter, and occurs without adversely affecting cell viability.