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[Construction of vectors encoding mouse Dishevelled 2 and its expression in RAW264.7 cells]
Xu Huang1, Juan Zhao, Ying-Jie Mao
1Dept. of Stomatology, The First Affiliated Hospital of Medical College of Zhejiang University, Hangzhou 310003, China.
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 significance of Dvl2 in the osteoclastogenesis.
