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: Laura H R Leite, Neeru M Sharma, Sangeeta Bafna, Hong Zheng, Cândido C Coimbra, Kaushik P Patel
• Journal: Journal of neuroscience methods (J Neurosci Methods) Vol. 211 Issue 1 Pg. 77-83 (Oct 15 2012) ISSN: 1872-678X [Electronic] Netherlands
• PMID: 22921486 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2012 Elsevier B.V. All rights reserved.

Chemical References

• enhanced green fluorescent protein
• Green Fluorescent Proteins
• Nitric Oxide Synthase Type I
• NADPH Dehydrogenase

Topics

• Animals
• Blotting, Western
• Cell Line
• Fluorescent Antibody Technique
• Gene Expression Regulation, Enzymologic
• Gene Transfer Techniques
• Genetic Vectors
• Green Fluorescent Proteins (genetics)
• Humans
• Immunohistochemistry
• Lentivirus (genetics)
• Microinjections
• Midline Thalamic Nuclei
• NADPH Dehydrogenase (metabolism)
• Neurons (physiology)
• Nitric Oxide Synthase Type I (biosynthesis, genetics)
• Rats
• Real-Time Polymerase Chain Reaction
• Reproducibility of Results