[Construction of the transfected cell line expressing the human CXCR4 gene and study of its biological function]
Shi-yong Zhou1, Ming-feng Yang, Xue-feng Wang
1Biotechnology Institute of Soochow University, Key Labatory of Stem Cell of Jiangsu Province, Suzhou 215007, China. aiwintersweet@yahoo.com.cn
Aim:
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.
Conclusion:
The CXCR4 transfected L929 cell line was successfully established, and it can make the basis for the further research.
