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Updated: Aug 9, 2026
Inducible and Reversible Dominant-negative (DN) Protein Inhibition
Published on: January 7, 2019
[Construction of Tet-on inducible CXCR1 eukaryotic expression plasmid and identification of the expression character
Man Shi1, Li-bo Yao, Li-feng Wang
1Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an 710032, China.
Aim:
To construct tetracycline (Tet)-controlled inducible vector CXCR1-pTREhyg, and then detect the expression character of CXCR1 under the regulation of Dox in NIH3T3 cells.
Methods:
A full length cDNA of human CXCR1 was cloned from sample of fibroblast like synovium (FLS) in rheumatoid arthritis (RA) patient by RT-PCR and then sub-cloned into the pTREhyg plasmid after sequence analysis. CXCR1-pTREhyg and pTet-on was co-transfected with Lipofect2000 to NIH3T3 cells, and the expression of IL-8RA was detected by Western blot after given different concentration of Dox.
Results:
Western blot showed that CXCR1 could be induced by Dox in NIH3T3 cells, and the phosphorylated Erk-1/2 level was significantly increased after IL-8 stimulation.
Conclusion:
Tet inducible recombinant vector of CXCR1-pTREhyg was successfully constructed, and it could be expressed in NIH3T3 cells. The stimulation of IL-8 obviously changed the activity of Erk-1/2 in the transfected NIH3T3 cells. This work has laid foundations for further study on the relationship between CXCR1 and RA disease.
