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 × 10⁴ pfu/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.

Figure 1

Real time PCR analysis of nNOS in NG108 cells treated with 3 concentrations of lenti-EGFP and lenti-nNOS for 24h. Values are mean±SE of three different experiments. * Significantly different from control without any treatment; # Significantly different from corresponding dose between different groups, P<0.05.

Figure 2

nNOS expression in NG108 cells treated with 3 concentrations of lenti-EGFP and lenti-nNOS for 24h. Top: a representative Western blot, bottom: densitometric analyses of nNOS level normalized to GAPDH. Values are mean±SE of four different experiments. * Significantly different from control without any treatment; # Significantly different from corresponding dose between different groups, P<0.05.

Figure 3

Representative immunofluorescent photomicrographs of Lenti-EGFP (A) and Lenti-nNOS (B) (2.5× 10⁴ pfu/ml) treated NG108 cells for 24h and stained for nNOS (green). Blue spots show the nucleus stained by 4′, 6′-diamidino-2-phenylindole (DAPI)

Figure 4

Time course of nNOS in lenti-EGFP-treated (A) and lenti-nNOS-treated (B) NG108 cells after 1, 2 and 3 weeks. Top: a representative Western blot, bottom: densitometric analyses of nNOS level normalized to GAPDH. Values are mean±SE of four different experiments. * Significantly different from control without any treatment, P<0.05.

Figure 5

(A) Representative histochemical photomicrographs of NADPH-diaphorase labeled neurons (blue staining) in the PVN at similar coronal level. Left side: lenti-nNOS injected side. Scale bar=200μm. 3V: third ventricle. (B) Ratio of nNOS intensity in the PVN of two independent experiments. Values expressed as mean ± SEM, n=2 rats. NI: non-injected side; I: injected side.

Figure 6

Time course of nNOS in the PVN of rats microinjected unilaterally with lenti-EGFP and lenti-nNOS after 1, 2 and 3 weeks. Values expressed as mean ± SEM, n=4 rats. NI: non-injected side; I: injected side. * Significant difference from non-injected side within the same week and group; # Significant difference from corresponding PVN side and week between different groups, P<0.05.