[Generation and preliminary functional study of a CD133-2-harboring L929 cell line].

AIM

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.

CONCLUSION

The engineered CD133-2/L929 cell line provides a gain-of-function cell model for further understanding the biological role of CD133-2.