PMID: 16643778Apr 29, 2006Paper

Construction of Tet-on inducible CXCR1 eukaryotic expression plasmid and identification of the expression character in NIH3T3 cells

Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology
Man ShiXin-ping Liu

Abstract

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. 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. 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. 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.

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