[Generation and preliminary functional study of a CD133-2-harboring L929 cell line].
OBJECTIVE
To generate an engineered L929 cell line harboring human CD133-2 and perform the functional study of the gene-modified cell line.


METHODS
The human CD133-2 gene was obtained by PCR from a cDNA library of foetus liver. After digested with Hind III and BamH I, the PCR product was cloned into pIRES2-EGFP vector. The recombinant plasmid CD133-2/pIRES2-EGFP was transfected into L929 cell line using lipofectamine, followed by G418 selection. RT-PCR, Western blot and flow cytometry (FCM) were used to detect the expression of CD133-2. MTT was used to analyze the effect of the CD133-2/L929 cells on proliferation of T cells. FCM was performed to monitor T cell activation by detecting T cell surface markers of CD4CD25 and CD8CD25 after T cells were cocultured with the CD133-2/L929 cells in the presence of an anti-CD3 mAb.


RESULTS
A stable cell line constitutively expressing the human CD133-2 was established successfully. In the presence of the anti-CD3 mAb, CD133-2/L929 cells caused inhibition of T cell proliferation and down-regulation of the activation markers of CD4CD25 and CD8CD25 on T cells.


CONCLUSIONS
The engineered CD133-2/L929 cell line provides a gain-of-function cell model for further understanding the biological role of CD133-2.
Wang
Jia-min
JM
Biotechnology Institute, Soochow University, Suzhou 215007, China. wangjianmin@suda.edu.cn
Chen
Yong-jing
YJ
Zhang
Guang-bo
GB
Li
Fang
F
Hu
Yu-min
YM
Zhang
Xue-guang
XG
D000071916:AC133 Antigen; D015703:Antigens, CD / Q000235:genetics / Q000276:immunology*; D002460:Cell Line / Q000276:immunology*; D049109:Cell Proliferation; D002478:Cells, Cultured; D003001:Cloning, Molecular; D015536:Down-Regulation; D015870:Gene Expression; D005818:Genetic Engineering; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology*; D006801:Humans; D010455:Peptides / Q000235:genetics / Q000276:immunology*; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology; D014162:Transfection
D000071916:AC133 Antigen; D015703:Antigens, CD; D006023:Glycoproteins; C000606023:PROM1 protein, human; D010455:Peptides