Identification and expression of shRNA vectors targeting human AMPKα2.
- Author:
Jun LU
1
;
Shi-yuan XU
;
Rui CUI
;
Qing-guo ZHANG
;
Hong-yi LEI
Author Information
- Publication Type:Journal Article
- MeSH: AMP-Activated Protein Kinases; genetics; Cell Line; Gene Targeting; methods; Genetic Vectors; genetics; Humans; RNA Interference; RNA, Small Interfering; genetics; metabolism; Transfection
- From: Journal of Southern Medical University 2011;31(1):86-89
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct pGPU6/GFP/Neo-shRNA expression vector targeting human AMPKα2 gene and evaluate its silencing effect in SH-SY5Y cell line.
METHODSThe 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.
RESULTSFour 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.
CONCLUSIONTransfection 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.
