| Objective To construct and identify recombinant adenovirusesAd-IRF8-TRAPpromoter and Ad-IRF8-CMV and observe the influence ofthe over-expressed IRF8on the differentiation of osteoclast precursorcells(RAW264.7) into osteoclast-like cells induced by soluble receptoractivator of nuclear factor κB ligand (sRANKL).Methods Mouse IRF8gene and TRAP promoter amplified by PCRwere subcloned into the shuttle vector pAdTrack. In addition, Mouse IRF8gene amplified by PCR was subcloned into pAdTrack-CMV. The vectorspAdTrack-IRF8-TRAPpromoter and pAdTrack-IRF8-CMV were linearizedby PmeⅠ,followed by homologous recombination with bone vectorpAdEasy-1in E.coli BJ5183. Recombinant adenovirus vectorspAd-IRF8-TRAPpromoter and pAd-IRF8-CMV were obtained andlinearized by PacⅠ,and then transfected into AD293cells.The recombinantadenoviruses Ad-IRF8-TRAPpromoter and Ad-IRF8-CMV were harvestedafter packaged in AD293cells. RAW264.7cells were infected byAd-IRF8-TRAPpromoter and Ad-IRF8-CMV.The overexpression of IRF8 was verified with semi-quantitative reverse transcription-PCR(RT-PCR)and Western blotting. The influence of the overexpression of IRF8onsRANKL-induced differentiation of RAW264.7cells was detected bytartrate-resistant acid phosphatase (TRAP) staining.Results The shuttle vector pAdTrack-IRF8-TRAPpromoter/CMV andrecombinant vector pAd-IRF8-TRAPpromoter/CMV were successfullyconstructed.The recombinant adenovirus Ad-IRF8-TRAPpromoter/CMVwas also successfully packaged in AD293cells.The overexpression of IRF8in RAW264.7cells was confirmed by RT-PCR and Western blotting.TRAPstaining showed that the overexpression of IRF8effectively inhibited thedifferentiation of RAW264.7cells into osteoclast-like cells.Conclusion The recombinant adenoviruses Ad-IRF8-TRAPpromoterand Ad-IRF8-CMV were successfully constructed and the overexpressionof IRF8was also detected in RAW264.7cells.The over-expressed IRF8caneffectively inhibit the differentiation of RAW264.7cells into osteoclast-likecells．... |
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