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Construction and Selection of siRNA Plasmid Vector for Suppressing Coronin-1 Gene Expression

Author: YanSha
Tutor: ChenQuan
School: Chongqing Medical University
Course: Medical Immunology
Keywords: Coronin-1 RNA interference siRNA plasmid vector Mycobacterium tuberculosis
CLC: R346
Type: Master's thesis
Year: 2010
Downloads: 20
Quote: 0
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Abstract


Objective: To construct specific inhibition of gene expression Coronin-1 siRNA expression vector and screened with the highest inhibition efficiency of the plasmid vector . Methods: mouse macrophages extracted total RNA, RT-PCR amplification of target sequences Coronin-1 gene , and connect it to pSEB-HUS eukaryotic expression plasmid , the recombinant plasmid by restriction analysis and DNA sequencing named after pSEB-HUS -C. The design, synthesis of 3 and negative control siRNA were cloned into pSEB-HUS-C, recombinant pSEB-HUS-C1, pSEB-HUS-C2, pSEB-HUS-C3 and pSEB-HUS-CN plasmid . Would interfere with A549 cells were transiently transfected by green fluorescence signal observed on different recombinant Coronin-1 expression. Finally, quantitative real-time PCR and Western blot assay its Coronin-1 gene expression inhibition efficiency . Results: After double digestion and sequencing confirmed that the constructed siRNA expression vector gene size, serial line with expectations. The transiently transfected A549 cells, which pSEB-HUS-C3 could inhibit Coronin-1 mRNA expression and Coronin-1 protein synthesis , inhibition rates were 75.9% and 75.1% . Conclusion : We successfully constructed and screened efficient, specific inhibition of Coronin-1 expression by siRNA expression vector for further study Coronin-1 in macrophages role foundation.

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