Inhibition of HCV IRES controlled reporter gene expression by RNA interference / 中华肝脏病杂志
Chinese Journal of Hepatology
; (12): 521-524, 2006.
Article
em Zh
| WPRIM
| ID: wpr-341319
Biblioteca responsável:
WPRO
ABSTRACT
<p><b>OBJECTIVE</b>To develop a RNAi approach that specifically targets the HCV IRES sequence by vector-expressed short hairpin RNA (shRNA) in vitro, and to assess the inhibitory effect of the shRNA on reporter gene expression.</p><p><b>METHODS</b>Eukaryotic expressing plasmids, pIRES-GFP and p5' UTR-Luc containing GFP or luciferase gene controlled by HCV IRES were cotransfected into HepG2 cells with either a RNAi plasmid pshRNA-HCV or a control plasmid pTZU6+1. At 24, 48, 72 hours post transfection, the fluorescence in the transfected cells was studied using fluorescence microscopy. The levels of GFP RNA were determined using RT-PCR and those of protein were determined using Western blot. The activities of luciferase were assayed using a dual luciferase assay system.</p><p><b>RESULTS</b>The introduction of RNAi plasmid efficiently and specifically down-regulated the expression of the reporter gene. RT-PCR showed that the RNAs of GFP gene were distinctly reduced (about 60%) when the pIRES-GFP was cotransfected with pshRNA-HCV, whereas the control vector did not exhibit inhibitory effect on the mRNA level, according to Western blot assay. The luciferase activity also decreased by 60%-70% in comparison to the control plasmid.</p><p><b>CONCLUSION</b>Our results demonstrate that the shRNA targeting HCV IRES shows a strong inhibitive effect on the expression of the reporter gene controlled by this sequence, suggesting that RNAi-based anti-HCV strategy may represent a potential approach in the therapy of HCV infection.</p>
Texto completo:
1
Índice:
WPRIM
Assunto principal:
Ribossomos
/
Terapêutica
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RNA Mensageiro
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Transfecção
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Terapia Genética
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Regulação da Expressão Gênica
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Hepatite C
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Genes Reporter
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Hepacivirus
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Interferência de RNA
Limite:
Humans
Idioma:
Zh
Revista:
Chinese Journal of Hepatology
Ano de publicação:
2006
Tipo de documento:
Article