小鼠Dishevelled 2表达质粒的构建及其在RAW264.7细胞中的表达

Construction of vectors encoding mouse Dishevelled 2 and its expression in RAW264.7 cells

目的构建小鼠Dishevelled 2(Dvl2)表达质粒,并检测其转染小鼠破骨前体细胞RAW264.7后的表达,为进一步研究Dvl2在破骨细胞分化和骨重建中的作用、筛选调节骨重建潜在靶点奠定基础。方法根据GenBank小鼠Dvl2 cDNA序列设计两条特异性引物,提取RAW264.7细胞总RNA,以RT-PCR获得Dvl2的开放阅读框(ORF),并将其克隆到真核表达质粒pEZ-M29上,酶切电泳,测序鉴定构建的pEZ-M29/Dvl2质粒。用脂质体介导瞬时转染RAW264.7细胞,通过荧光显微镜观察转染效果,实时定量RT-PCR检测Dvl2基因表达情况。结果成功构建pEZ-M29/Dvl2表达载体,酶切电泳和测序结果与目的基因相符;转染RAW264.7细胞后48 h,荧光显微镜下可见绿色荧光蛋白表达;实时定量RT-PCR可见实验组Dvl2基因较对照组强烈过表达。结论成功构建小鼠Dvl2真核表达质粒pEZ-M29/Dvl2,瞬时转染小鼠破骨前体细胞RAW264.7可显著提高Dvl2的mRNA水平。

Objective To construct the recombinant vectors that express mouse Dishevelled 2（Dvl2）,and to evaluate its expression level in transfected RAW264.7 cells.Methods A pair of specific primers were designed according to the mouse Dvl2 cDNA sequence published in GenBank.Total RNA of RAW264.7 cells was extracted,and open reading frame of Dvl2 was obtained by RT-PCR,which was then cloned into pEZ-M29 plasmid.Electrophoresis after macrorestriction and DNA sequence analysis were used to identify the reconstructed plasmids.After transient transfection via liposome,the transfection of RAW264.7 cells was confirmed under a fluorescence microscope,and the expression level of Dvl2 was evaluated by real-time RT-PCR.Results The recombinant plasmid containing mouse Dvl2,namely pEZ-M29/Dvl2,was successfully constructed,and confirmed by DNA sequence analysis.After 48 h of tranfection,the expression of enhanced green fluorescene protein was observed under a fluorescence microscope,and real-time RT-PCR analysis revealed that the Dvl2 mRNA level was prominantly elevated in the transient transfected RAW264.7 cells.Conclusion The recombinant plasmids pEZ-M29/Dvl2 are successfully constructed and can elevate the Dvl2 mRNA level in the transient transfected RAW264.7 cells,which can be used in further studies aiming at revealing the functional signifi-cance of Dvl2 in the osteoclastogenesis.