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


CONCLUSIONS
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.
Shi
Man
M
Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an 710032, China.
Yao
Li-bo
LB
Wang
Li-feng
LF
Li
Fu-yang
FY
Su
Jin
J
Zhang
Jian
J
Li
Xia
X
Liu
Xin-ping
XP
D000818:Animals; D002478:Cells, Cultured; D004790:Enzyme Induction / Q000235:genetics; D005057:Eukaryotic Cells; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D041681:NIH 3T3 Cells; D010957:Plasmids / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology*
D013752:Tetracycline