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Updated: Jun 14, 2026
Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System
Published on: December 5, 2020
[Construction of a siRNA plasmid for knockdown of coronin-1]
Sha Yan1, Quan Chen, Yu-chun Lü
1Department of Immunology, Chongqing Medical University, Chongqing 400016, China. sinc555@126.com
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.
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