[Construction of the transfected cell line expressing the human CXCR4 gene and study of its biological function].
OBJECTIVE
To construct the tranfected cell line expressing the human CXCR4 gene and to study the biological function.


METHODS
The total RNA was isolated from peripheral blood mononuclear cell (PBMC) with TRIzol, and the CXCR4 gene was amplified by RT-PCR, then digested with restriction endonuclease Pst I and EcoR I, and inserted into retrovirus vector pEGZ-Term. The recombinant vector together with its two helper virus vectors was co-transfected into the package cells 293T with LipofectAMINE 2000. Then the supernatant of the 293T cell culture was used to infect L929 cells, the cell clones stably expressing the CXCR4 molecule were screened in the presence of Zeocin (500 mg/L) after 72 h cultivation.


RESULTS
It was found that the full-length of CXCR4 gene was successfully cloned, and the recombinant retrovirus vector carrying the CXCR4 gene was constructed. The CXCR4 cDNA transfected L929 cell could stably express the human CXCR4 on the cell membrane, and the migration ability of transfected cells was well evidenced in the transwell system induced by SDF-1alpha after the transfection with CXCR4.


CONCLUSIONS
The CXCR4 transfected L929 cell line was successfully established, and it can make the basis for the further research.
Zhou
Shi-yong
SY
Biotechnology Institute of Soochow University, Key Labatory of Stem Cell of Jiangsu Province, Suzhou 215007, China. aiwintersweet@yahoo.com.cn
Yang
Ming-feng
MF
Wang
Xue-feng
XF
Yu
Ge-hua
GH
Cheng
Zhong-qin
ZQ
Zhang
Xue-guang
XG
D000818:Animals; D045744:Cell Line, Tumor; D002465:Cell Movement; D054377:Chemokine CXCL12 / Q000378:metabolism; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D019718:Receptors, CXCR4 / Q000096:biosynthesis* / Q000235:genetics*; D014162:Transfection / Q000379:methods*
C527276:CXCR4 protein, human; D054377:Chemokine CXCL12; D019718:Receptors, CXCR4; D004262:DNA Restriction Enzymes