Objective: To construct pGPU6/GFP/Neo-shRNA expression vector targeting human AMPKα2 gene and evaluate its silencing effect in SH-SY5Y cell line.
Methods: 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.
Results: 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.
Conclusion: 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.