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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