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Cis-Reporting System Monitors Intracellular Calcium Mobilization

human T-cell line, or CHO cells were transfected with the pNFAT-Luc plasmid and treated with 60 ng/ml of PMA and 1g/ml of inomycin, luciferase activity increased by 13- and 16-fold, respectively (Figure 2). Therefore, the enhancer element in the pNFAT-Luc plasmid is responsive to calcium mobilization. Cells transfected with pNFAT-Luc and then treated with either PMA or ionomycin alone did not show a significant increase in luciferase expression (result not shown), which is consistent with previous reports.2

Fig.2

Cyclosporin is an immunosuppressive drug that was introduced in 1983 and revolutionized transplant surgery. It works primarily by inhibiting the activity of calcineurin, a protein phosphatase regulated by intracellular calcium mobilization. All the isoforms of NFAT protein contain a calcineurin-binding domain and are activated by calcineurin. Results in Figure 2 demonstrated that the luciferase expression from pNFAT-Luc in both Jurkat and CHO cells induced by PMA and ionomycin could be completely inhibited by cyclosporin. In another set of experiments, rat basophilic leukemia cells stably transfected with chemokin receptors were transfected with pNFAT-Luc and then treated with their respective ligands (data not shown). When both luciferase expression and calcium levels were monitored in these cells, luciferase expression correlated very well with calcium mobilization. Therefore, luciferase expression from pNFAT-Luc indeed reflects the activation of endogenous NFAT proteins by calcium immobilization.

Conclusions

The pNFAT-Luc plasmid is a new addition to Stratagenes PathDetect cis-reporting systems. Stratagene has developed
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