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- W2391831443 abstract "Objectives To construct a novel retroviral vector delivery shRNA based on the Pmscv/Hyg vector,and investigate its effect of gene silencing on HEK293 cells.Methods Human U6 promoter containing the introduced BamHI and SalI sites was amplified from genomic DNA and reversely inserted into the upstream of the 3'LTR in Pmscv/Hyg.The EGFP gene was cloned from plasmid PEGFP-N1 and inserted into the multiclone sites in Pmscv/Hyg.The oligonucleotides encoding the human P53 siRNA was annealed and ligated into the BamHI and SalI sites. After screening and amplification,the recombinant retroviral plasmid was obtained and transfected into PT67 cells.The HEK293 cells were transduced with retrovirus supernatant.The positive clones were selected by Hygromycin.The expression of P53 mRNA and protein were evaluated by RT-PCR and Western blotting respectively.Results The novel recombinant vector was confirmed by agarose gel electrophoresis after restriction enzyme digestion and by sequence analysis.RT-PCR and Western blot analysis demonstrated a remarkable downregulation of P53 mRNA and protein in HEK293 cells infected with Pmscv/P53shRNA.Conclusion The retroviral vector Pmscv/EGFP/shRNA has been successfully constructed,and he retroviral system is capable of delivering short interference RNA against target gene." @default.
- W2391831443 created "2016-06-24" @default.
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- W2391831443 date "2009-01-01" @default.
- W2391831443 modified "2023-09-24" @default.
- W2391831443 title "Construction and Identification of a Novel Retroviral Vector Delivery shRNA based on the Pmscv/Hyg" @default.
- W2391831443 hasPublicationYear "2009" @default.
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