Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences · 2014
[Construction and evaluation of human Dp71 shRNA vector].
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Tan Sichuang, Chen Zhikang, Cao Xiaoxia, Wen Qiaocheng, Zhang Weilin, Zeng Qingren, Tan Sipin
Аннотация
OBJECTIVE: To construct effective short hairpin RNA (shRNA) recombinant plasmids targeting human Dystrophin Dp71 gene, and evaluate their interference efficiency.
METHODS: Three pairs of siRNA sequences targeting human Dp71 gene and one pair of control siRNA sequence were designed, synthesized, and then inserted into the pRNAT-U6.1/Neo vector. The shRNA recombinant vectors were evaluated by enzyme digestion and sequencing. Dp71-shRNA and control shRNA plasmids were transfected into human normal gastric epithelial cells (GES-1) and human bronchial epithelium (HBE). Western blot was used to evaluate its interfering efficiency.
RESULTS: Restriction enzyme digestion and sequencing showed that the Dp71-shRNA vectors were successfully constructed. Western blot displayed that Dp71 protein expression was reduced to a significant degree after transfection with the 3 Dp71-shRNA plasmids, and Dp71-shRNA2 plasmid inhibit the Dp71 expression most efficiently.
CONCLUSION: Dp71-shRNA vectors have been successfully constructed. The 3 Dp71-shRNA plasmids can inhibit Dp71 expression in GES-1 and HBEC, with Dp71-shRNA2 plasmid displaying the highest inhibition efficiency.
METHODS: Three pairs of siRNA sequences targeting human Dp71 gene and one pair of control siRNA sequence were designed, synthesized, and then inserted into the pRNAT-U6.1/Neo vector. The shRNA recombinant vectors were evaluated by enzyme digestion and sequencing. Dp71-shRNA and control shRNA plasmids were transfected into human normal gastric epithelial cells (GES-1) and human bronchial epithelium (HBE). Western blot was used to evaluate its interfering efficiency.
RESULTS: Restriction enzyme digestion and sequencing showed that the Dp71-shRNA vectors were successfully constructed. Western blot displayed that Dp71 protein expression was reduced to a significant degree after transfection with the 3 Dp71-shRNA plasmids, and Dp71-shRNA2 plasmid inhibit the Dp71 expression most efficiently.
CONCLUSION: Dp71-shRNA vectors have been successfully constructed. The 3 Dp71-shRNA plasmids can inhibit Dp71 expression in GES-1 and HBEC, with Dp71-shRNA2 plasmid displaying the highest inhibition efficiency.