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Stable HeLa Luciferase Reporter Cell Lines Expressing GAL4 Fusion,,,Transactivators

porter cell lines that express the GAL4-CREB, GAL4-Elk1, or GAL4-cJun. In the HLR-CREB, HLR-Elk1, and HLR-cJun cell lines, luciferase activity reflects the activation status of the chimeric trans-activator protein and, therefore, the respective signaling pathways. By performing simple luciferase assays, researchers can directly evaluate the effects of extracellular stimuli on signaling pathways converging at CREB, Elk1, or c-Jun. Use of these cell lines eliminates transfections, thereby saving time while avoiding the variables that can be caused by fluctuations in transfection efficiencies. The HLR cell line can be used to assess protein-protein interactions in mammalian cells,11 while the HLR-CREB, HLR-Elk1, and HLR-cJun cell lines can be used to study transcription mechanisms. Another application for these cells includes adaptation for high-throughput drug screenings, since many components of signal transduction pathways are potential targets for drug development efforts.

Establishing Stable Reporter Cell Lines

Fig.1

To create stable reporter cell lines with the PathDetect trans-reporting systems, we tried several approaches. Figure 1 outlines our most successful protocol: We first transfected the pFR-Luc plasmid with a hygromycin expression cassette into HeLa cells. Through successive rounds of selection in antibiotic medium, hygromycin-resistant clones were isolated and tested with the PathDetect CREB, Elk1, and c-Jun trans-reporting systems. Cell lysates from these stable clones were then prepared, assayed for luciferase activity, and analyzed. One clonal line, designated as HLR for the HeLa luciferase reporter cell line, was chosen for further experimentation. It gave the highest levels of luciferase activity when tested with the trans-reporting sys
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