1. [Construction of a siRNA plasmid for knockdown of coronin-1].
- Author
-
Yan S, Chen Q, Lü YC, Hou Y, and Wang YW
- Subjects
- Animals, Blotting, Western, Cell Line, Tumor, Humans, Mice, Microfilament Proteins metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, Recombinant Fusion Proteins deficiency, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Reverse Transcriptase Polymerase Chain Reaction, Gene Knockdown Techniques methods, Microfilament Proteins deficiency, Microfilament Proteins genetics, Plasmids genetics, RNA, Small Interfering genetics
- Abstract
Aim: To construct a siRNA plasmid to knockdown Coronin-1., Methods: The cDNA of coronin-1 was amplified by RT-PCR from the total RNA of macrophage, and then inserted into pSEB-HUS vector to generate pSEB-HUS-C plasmid. Three synthesized siRNAs targeting Coronin-1 were cloned into pSEB-HUS-C respectively, resulting in the pSEB-HUS-C1, pSEB-HUS-C2 and pSEB-HUS-C3 plasmids. These plasmids were transiently transfected into A549, and the Coronin-1 level was detected by RT-PCR, Real time PCR and Western blot., Results: All plasmids were successfully constructed as confirmed by restriction enzyme digestion and DNA sequencing. The pSEB-HUS-C3 vector had the most significant knockdown effect on Coronin-1, with 75.9% inhibition at mRNA level and 75.1% inhibition at protein level., Conclusion: A siRNA plasmid targeting Coronin-1 was successfully constructed and validated for its knockdown effect, which will serve as a loss-of-function tool for the further mechanistic study of Coronin-1 in tuberculosis pathology.
- Published
- 2010