Dishevelled 2 siRNA载体的构建和有效干扰质粒的筛选

Construction of Dishevelled 2-targeted siRNA vectors and identification of the effective recombinant plasmids

目的构建和筛选特异性抑制Dishevelled 2(Dvl2)表达的siRNA载体,为进一步研究Dvl2在破骨细胞分化及骨重建中的作用、筛选调节骨重建潜在靶点而奠定基础。方法根据目的基因设计5个siRNA靶点,与pMAGic 4.0载体形成重组质粒,转化DH5α感受态细胞,含抗生素的LB琼脂平板上筛选转化子,菌落PCR鉴定阳性克隆,测序验证正确的转化子,质粒抽提,Lipofectamine 2000将质粒瞬时转染RAW264.7细胞,通过绿色荧光信号观察转染,实时定量RT-PCR检测Dvl2基因表达的变化。结果菌落PCR和测序证实所构建的siRNA质粒目的基因大小、序列与预期相符,能有效抑制RAW264.7细胞Dvl2基因的表达,其中3号质粒抑制效率最高。结论成功构建并筛选出特异性抑制Dvl2 mRNA表达的siRNA载体,可用于包装病毒颗粒和构建Dvl2表达抑制的稳定转染RAW264.7细胞。

Objective To construct Dishevelled 2（Dvl2）-targeted siRNA plasmids and to identify the effective recombinant plasmids in transciently-transfected RAW264.7 cells.Methods The interfering sequences of Dvl2 were de-signed according to the sequence of Dvl2 of GenBank.Five paires of oligonucleotides were synthesized and inserted into plasmid pMAGic 4.0 to generate siRNA expression vectors,which were identified by flora PCR and sequence analysis.The recombinant plasmids siRNA-Dvl2 was transciently transfected into RAW264.7 cells by Lipofectamine 2000,which was confirmed under a fluorescence microscope and the interfering efficiency was detected by real-time RT-PCR.Results Five Dvl2 siRNA frames were successfully inserted into the plasmid vector pMAGic 4.0,and the flora PCR and sequence analysis confirmed the correct construction.Three of the five siRNA vectors suppressed the expression of Dvl2 mRNA,in which the siRNA-Dvl2-3 was the most efficient.Conclusion The Dvl2-targeted recom-binant siRNA plasmids can be constructed successfully to inhibit Dvl2 mRNA expression in transciently-transfected RAW264.7 cells,which can be used to pack virus particles and to construct siRNA-Dvl2 stably-transfected RAW264.7 cells in further research.