Construction and validation of lentiviral vector carrying rat neuronal nitric oxide synthase in vitro and in vivo

Abstract

In the present study, we developed a lentiviral vector with human cytomegalovirus promoter permitting high-level of nNOS expression. Neuronal cell line NG108 was used as an in vitro model to check the validity of gene transfer. The cells were infected with lenti-EGFP or lenti-nNOS particles for 24h. Lenti-nNOS infection in the NG108 cells induced dose dependent increase in mRNA and protein for nNOS; with a dose of 2.5×104pfu/ml, nNOS mRNA expression increased by 40-fold while protein expression was increased by 2.5-fold compared to lenti-EGFP. Moreover, lenti-nNOS infection caused a greater increase in nNOS immunoreactivity in NG108 cells compared to lenti-EGFP as shown by immonocytochemistry. nNOS expression showed time dependent increases with lenti-nNOS infection with maximum up-regulation observed after two weeks of infection. Moreover, in vivo, unilateral injection of lenti-nNOS into the paraventricular nucleus (PVN) of rats induced a 27-fold increase of nNOS protein level in the injected side compared to non-injected side and this escalation was sustained up to three weeks. Overall, lenti-EGFP injection in the PVN did not show any significant change in nNOS expression. Furthermore, NADPH-diaphorase staining of nNOS in the PVN infected with lenti-nNOS induced a visible increase in nNOS expression compared with contralateral non-injected side up to three weeks. These results indicate that this approach of lentiviral mediated gene transfer of nNOS may provide a new means to up-regulate the nNOS expression for longer periods of time compared to adenoviral transfection and can be used as a research tool and potentially a therapy for chronic diseases involving impaired nNOS expression.