Abstract:

Objective To construct Dishevelled 2（Dvl2）-targeted siRNA plasmids and to identify the effective re- combinant 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.

Key words: Dishevelled 2, siRNA, RAW264.7 cell, osteoclast