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.
Article
в Zh
| WPRIM
| ID: wpr-267666
Ответственная библиотека:
WPRO
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>
Полный текст:
1
База данных:
WPRIM
Основная тема:
Transfection
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Cell Line
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Gene Targeting
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RNA, Small Interfering
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RNA Interference
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AMP-Activated Protein Kinases
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Genetic Vectors
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Genetics
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Metabolism
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Methods
Тип исследования:
Diagnostic_studies
Пределы темы:
Humans
Язык:
Zh
Журнал:
Journal of Southern Medical University
Год:
2011
Тип:
Article