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Cis-Reporting System Monitors Intracellular Calcium Mobilization

orting system includes the transfection-ready pNFAT-Luc reporter plasmid and the pCIS-CK negative control plasmid.

Construction of the pNFAT-Luc Plasmid

Fig.1

The backbone of the 5749-base-pair pNFAT-Luc plasmid (Figure 1) is the pFR-Luc reporter plasmid of the PathDetect trans-reporting system. To this backbone, we replaced the GAL4 binding element with four direct repeats of the NFAT binding sequence (286 to 257) from the IL-2 gene promoter, the most studied and widely used NFAT binding sequence.1,2,10,11 In addition to a polyadenylation signal downstream of the luciferase gene, the pNFAT-Luc plasmid contains another polyadenylation signal in front of the NFAT binding sites and TATA box. This design minimizes transcription read through from upstream sequences and background luciferase expression. For all reporter plasmids of the PathDetect cis-reporting systems, activation of the luciferase gene indicates interaction of uncharacterized gene products, extracellular stimuli, growth factors, or drug candidates with specific enhancer elements. When a plasmid expressing the gene of interest is cotransfected into mammalian cells along with a cis-reporter plasmid, luciferase activity indicates transcription activation.

Testing the pNFAT-Luc Plasmid in Jurkat Cells

Under natural cellular conditions, NFAT proteins are activated by antigen stimulation of the T-cell receptor.1 Pharmacology studies have established that NFAT proteins can be activated by the protein kinase C activator phorbol ester (PMA) in combination with the calcium ionophore ionomycin, reagents that raise free intracellular calcium.2,12 When Jurkat cells, a mature
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