( eLa cells transfected with the pISRE-Lu...)New Reporter Plasmids for Studying Interferon-Stimulated Signal Transduction,,,Pathways

eLa cells transfected with the pISRE-Luc plasmid and treated with 200 to 10,000 U/ml of IFN-a 9- to 16-fold increase in luciferase activity was seen. As expected, IFN-b treatment of the cells also stimulated luciferase expression from the pISRE-Luc plasmid up to 11-fold (Figure 1, solid circles). Although the pISRE-Luc plasmid was somewhat responsive to IFN-g (Figure 1, solid triangles), the luciferase activity was considerably higher for HeLa cells treated with IFN-a and IFN-b (Figure 1, solid squares and solid circles). This weak response of the pISRE-Luc plasmid to IFN-g could be attributed to the similarity of the ISRE and GAS sequences; it is also likely that types I and II interferons use a few common components to transmit their signals to the cell nucleus. However, neither IFN-a nor IFN-b induced luciferase expression from HeLa cells transfected with the pCIS-CK plasmid, a control plasmid that differs from the cis-reporter plasmids only in that it lacks an enhancer sequence (Figure 1, open squares and open circles). We conclude that the pISRE-Luc reporter plasmid is effective for monitoring the signaling pathways activated by type I interferons.

Construction and Testing of the pGAS-Luc Plasmid

The human gene encoding guanylate-binding protein (GBP) is induced in fibroblasts by INF-g within 15 minutes of treatment. An IFN gamma-activating sequence (GAS) has been identified in the GBP promoter (nucleotides -123 to -103).¹ To construct the pGAS-Luc plasmid, we
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