title: 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.
authors.affiliation: Department of Physiology, Institute of Biological Sciences, Federal University of Juiz de Fora, 36036-900 Juiz de Fora, Minas Gerais, Brazil.
authors.forename: Laura H R
authors.initials: LH
authors.lastname: Leite
authors.forename: Neeru M
authors.initials: NM
authors.lastname: Sharma
authors.forename: Sangeeta
authors.initials: S
authors.lastname: Bafna
authors.forename: Hong
authors.initials: H
authors.lastname: Zheng
authors.forename: Cândido C
authors.initials: CC
authors.lastname: Coimbra
authors.forename: Kaushik P
authors.initials: KP
authors.lastname: Patel
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D015971:Gene Expression Regulation, Enzymologic; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D016086:Lentivirus / Q000235:genetics*; D008845:Microinjections; D020644:Midline Thalamic Nuclei; D009252:NADPH Dehydrogenase / Q000378:metabolism; D009474:Neurons / Q000502:physiology; D052248:Nitric Oxide Synthase Type I / Q000096:biosynthesis / Q000235:genetics*; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D052248:Nitric Oxide Synthase Type I; D009252:NADPH Dehydrogenase