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

Laura H.R. Leite, Neeru M. Sharma, Sangeeta Bafna, Hong Zheng, Cândido C. Coimbra, Kaushik P. Patel

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)77-83
Number of pages7
JournalJournal of Neuroscience Methods
Volume211
Issue number1
DOIs
StatePublished - Oct 15 2012

Fingerprint

Nitric Oxide Synthase Type I
Paraventricular Hypothalamic Nucleus
Infection
Up-Regulation
NADPH Dehydrogenase
Messenger RNA
Injections
Proteins
Cytomegalovirus
Genes
Transfection
Chronic Disease
Staining and Labeling
Cell Line
In Vitro Techniques
Research
Therapeutics

Keywords

  • Heart failure
  • Lentivirus
  • NNOS
  • PVN

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

Construction and validation of lentiviral vector carrying rat neuronal nitric oxide synthase in vitro and in vivo. / Leite, Laura H.R.; Sharma, Neeru M.; Bafna, Sangeeta; Zheng, Hong; Coimbra, Cândido C.; Patel, Kaushik P.

In: Journal of Neuroscience Methods, Vol. 211, No. 1, 15.10.2012, p. 77-83.

Research output: Contribution to journalArticle

Leite, Laura H.R. ; Sharma, Neeru M. ; Bafna, Sangeeta ; Zheng, Hong ; Coimbra, Cândido C. ; Patel, Kaushik P. / Construction and validation of lentiviral vector carrying rat neuronal nitric oxide synthase in vitro and in vivo. In: Journal of Neuroscience Methods. 2012 ; Vol. 211, No. 1. pp. 77-83.
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