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- W2348572827 abstract "Objective The ALKBH2 gene is highly expressed in urothelial tumors,and RNA interference( RNAi) technology cannot downregulate the target genes stably. The aim of this study was to construct and identify a lentiviral vector for RNA interference of human ALKBH2( ABH2). Methods The coding region of the ABH2 gene sequences were analyzed for a single-stranded oligo design and synthesis. PCR was performed for splicing reaction of synthetic oligo,which was synthesized into the pcDNA3. 1( +) vector and transformed into the competent cell DH5α. An expression vector pcDNA3. 1( +) / ABH2 was constructed after sequencing and identifying the gene sequence of the recombinant clones. According to the target gene sequence,three pairs of shRNA were designed and synthesized and built into the U6 carrier. The shRNA vectors were co-transfected with expression vectors into the HEK-293 cell line,and the optimal interference sequence was screened by QPCR. The screened shRNA-571,as the most effective interference sequence,was constructed into the lentiviral vector,pL / shRNA / F carrier. The 293T cells were co-transfected with the constructed lentiviral interference vectors and packaging plasmids,the viruses were packed,the virus stock was collected and concentrated by ultracentrifugation,and the titer of the viruses was determined. The LV-shRNA-57 was co-transfected with the over-expression vector pcDNA3. 1( +) /ABH2 into ACHN renal carcinoma,and the optimal interference sequence was screened by QPCR. Results Gene sequencing proved that the recombinant clone gene sequence was correct,and the lentiviral vector was successfully constructed. The titer of the concentrated virus was 1. 1 × 108TU / mL in the viral supernatant of 293T cells collected at 48 hours after co-transfection. The relative expression of ABH2 mRNA was significantly reduced in the siRNA-571 group after RNAi as compared with that in the control group( 0. 078 ± 0. 006 vs 0. 785 ± 0. 082,P 0. 01). The interference efficiency of the lentiviral vector PL / shRNA / F-ALKBH2-571 in the target gene ABH2 was 92. 2%. Conclusion A lentiviral RNAi expression vector targeting the ABH2 gene was successfully constructed and identified." @default.
- W2348572827 created "2016-06-24" @default.
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- W2348572827 date "2014-01-01" @default.
- W2348572827 modified "2023-09-23" @default.
- W2348572827 title "Construction and identification of a lentiviral RNAi expression vector targeting the ALKBH2 gene" @default.
- W2348572827 hasPublicationYear "2014" @default.
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