[Construction of a siRNA plasmid for knockdown of coronin-1].
OBJECTIVE
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.


CONCLUSIONS
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.
Yan
Sha
S
Department of Immunology, Chongqing Medical University, Chongqing 400016, China. sinc555@126.com
Chen
Quan
Q
Lü
Yu-chun
YC
Hou
Yan
Y
Wang
Yu-wei
YW
D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D055785:Gene Knockdown Techniques / Q000379:methods*; D006801:Humans; D051379:Mice; D008840:Microfilament Proteins / Q000172:deficiency* / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D008840:Microfilament Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C072061:coronin proteins