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Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology

[Construction of lentivirus-mediated short hairpin RNA targeting human STAT3 gene].


PMID 23127415

Abstract

To construct and identify a lentivirus-mediated short hairpin RNA (shRNA) targeting human signal transducer and activator of transcription 3 (STAT3) gene. The shRNA chains targeting to human STAT3 gene were designed and synthesized, and then inserted into lentivirus expression vector pSicoR containing U6 promoter and green fluorescent protein (GFP) gene by gene recombination technique. The constructed recombinant plasmid pSicoR-STAT3-shRNA was identified by double restriction enzyme digestion and DNA sequencing, and then mixed with the 3rd generation of lentiviral packaging system and co-transfected to HEK293 cells using new generation of Roche X-tremeGENE HP DNA Transfection Reagent mediated transfection method. RT-PCR and Western blotting were employed to detect the expressions of STAT3 at mRNA and protein levels, respectively. Negative plasmid transfected into the same cell line was used as a control group. Restriction analysis and sequencing proved that the recombinant plasmid pSicoR-STAT3-shRNA was constructed correctly. Lentivirus particles were successfully packaged in HEK293 cells with high titer. The expressions of STAT3 at mRNA and protein levels in the transfected HEK293 cells were weaker than those of the control group (P<0.05). The lentivirus-mediated shRNA targeting human STAT3 gene is successfully constructed.