Inducible expression of Mac-1-FP in Chinese hamster ovary cells using Tet-On gene expression system / 第二军医大学学报

ABSTRACT

Objective: To construct eukaryotic inducible expression plasmids pTRE-Tight-CFP-CD11b and pTRE-Tight-YFP-CD18 using Tet-On gene expression system and co-transfect them into Chinese hamster ovary (CHO) cells, so as to achieve the inducible expression of Mac-1-FP. Methods: The eukaryotic expression vectors pTRE-Tight-CFP-CN11b and pTRE-Tight-YFP-CD18 were constructed by recombinant DNA technique. The 2 vectors were co-transfected into CHO cells using liposome to fuse CD11b and CD18 the 2 subunits of Mac-1. Fluorescence microscope was used to observe the cyan fluorescence and the yellow fluorescence of Mac-1-FP. The influence of different levels of Dox (0, 0.01, 0.1, 0.5, 1, 2 μg/ ml) on expression levels of CD11b and CD18 in CHO cells was analyzed by RT-PCR and fluorescence intensity analysis. The adhesive rate of CHO-Mac-1-FP cells with ligand ICAM-1 was analyzed before and after PMA (1 μg/ml) stimulation. Results: The recombinant plasmids of pTRE-Tight-CFP-CD11b and pTRE-Tight-YFP-CD18 were successfully constructed. The cyan and yellow fluorescences were observed in co-transfected CHO cells under fluorescence microscope. The fluorescence intensity of the cells was increased with the increase of Dox concentration. RT-PCR analysis demonstrated that the CD11b and CD18 mRNA increased with the increase of Dox concentration. The adhesive rate of CHO-Mac-1-FP cells with ICAM-1 was increased after PMA stimulation (peaked at 2 h and 4 h after stimulation and decreased afterwards). Conclusion: This study achieves the inducible expression of Mac-1-FP in CHO cells. And Mac-1-FP, like widetype Mac-1, exhibits adhesive activity with ligand ICAM-1, which lays a foundation for studying the consisting dimmer, clustering, conformation and affinity of the ligands of Mac-1 using single molecule spectroscopy and fluorescence resonance energy transfer technique in living cells.