Identification and expression of shRNA vectors targeting human AMPKα2 / 南方医科大学学报
Journal of Southern Medical University
;
(12): 86-89, 2011.
Artigo
em Chinês
| WPRIM
| ID: wpr-267666
ABSTRACT
<p><b>OBJECTIVE</b>To construct pGPU6/GFP/Neo-shRNA expression vector targeting human AMPKα2 gene and evaluate its silencing effect in SH-SY5Y cell line.</p><p><b>METHODS</b>The oligonucleotides designed by Ambion online CAD software targeting AMPKα2 were cloned into the pGPU6/GFP/Neo vector. After confirmation by DNA sequencing and enzyme digestion analysis, the recombinant vectors were transfected into the SH-SY5Y cell line via lipofectamine and the positive clones were selected using G418. The expression levels of AMPKα2 mRNA and protein in the transfected cells were detected by RT-PCR and Western blotting, respectively.</p><p><b>RESULTS</b>Four shRNA vectors were successfully constructed as confirmed by DNA sequencing and the enzyme digestion analysis. Among the 4 recombinant vectors, pGPU6/GFP/Neo-shRNA AMPKα2(3) showed the strongest gene silencing effect and down-regulated the protein expression of AMPKα2 by 63% in the transfected cells.</p><p><b>CONCLUSION</b>Transfection with pGPU6/GFP/Neo-shRNA AMPKα2(3) results in effective inhibition of AMPKα2 gene expression in SH-SY5Y cells, which provide a means for studying AMPK-mediated cell injury.</p>
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Transfecção
/
Linhagem Celular
/
Marcação de Genes
/
RNA Interferente Pequeno
/
Interferência de RNA
/
Proteínas Quinases Ativadas por AMP
/
Vetores Genéticos
/
Genética
/
Metabolismo
/
Métodos
Tipo de estudo:
Estudo diagnóstico
Limite:
Humanos
Idioma:
Chinês
Revista:
Journal of Southern Medical University
Ano de publicação:
2011
Tipo de documento:
Artigo
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