title: [Identification and expression of shRNA vectors targeting human AMPKα2].
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Anesthesiology, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Lu
authors.forename: Shi-yuan
authors.initials: SY
authors.lastname: Xu
authors.forename: Rui
authors.initials: R
authors.lastname: Cui
authors.forename: Qing-guo
authors.initials: QG
authors.lastname: Zhang
authors.forename: Hong-yi
authors.initials: HY
authors.lastname: Lei
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000235:genetics*; D002460:Cell Line; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: D034741:RNA, Small Interfering; C526181:PRKAA2 protein, human; D055372:AMP-Activated Protein Kinases