Construction of shRNA of fulminant hepatitis related gene mfgl2 and investigation of its biological effects in vitro |
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Authors: | Dong Xi Zhi-Mo Wang Sui Gao Chuan-Long Zhu Jian-Wen Guo Xiao-Ping Luo Qin Ning |
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Affiliation: | Laboratory of Infectious Immunology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China |
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Abstract: | This study was designed to explore the RNA interference technique in inhibition of the expression of the mouse fibrinogen like protein 2 (mfgl2), which has been reported to be involved in the development a variety of diseases including fulminant viral hepatitis. A plasmid named p-mfgl2shRNA, complementary to the sequence of mfgl2 was constructed, while another short hairpin RNA (shRNA) which was a mutated form of the mfgl2shRNA sequences was used as a control. A plasmid named pEGFP-mfgl2 expressing the mfgl2-EGFP fusion protein was also constructed for the screening of the effect of p-mfgl2shRNA on mfgl2 expression. By cotransfection of p-mfgl2shRNA and pEGFP-mfgl2 or pcDNA3.1-mfgl2 expression construct into CHO cells or HeLa cells, the inhibition of mfgl2 expression by mfgl2shRNA was analyzed by direct observation through fluorescent microscopy, FACS, RT-PCR and immunohistochemistry staining. The experiments showed the significant inhibitory effect of p-mfgl2shRNA on mfgl2 expression at 48h post-transfection in both CHO and Hela cell lines with the inhibitory efficiency as high as 80.1%. The study demonstrated that the construct of p-mfgl2shRNA successfully interfered with the mfgl2 expression in vitro. These authors contributed equally. |
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Keywords: | Fulminant hepatitis fgl2 prothrombinase RNA interference shRNA |
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