Stable luciferase reporter cell lines for signal transduction pathway readout using GAL4 fusion transactivators

While GAL4 fusion activators have been widely used for dissecting signal tr ansduction pathways in transient assays, there has been surprisingly little reported on utilizing cell lines with stably integrated fusion activators. To avoid problems with the efficiency and reproducibility inherent to tran sient transfection, we describe here the generation and characterization of HeLa reporter cell lines, which contain a stably integrated luciferase gen e responsive to stably integrated and constitutively expressed GAL4-CREB or GAL4-Elk1 fusion activators. These cell lines exhibited extremely low basa l luciferase expression but robust response to various extracellular stimul i or the expression of signaling molecules that resulted in elevated MAP ki nase or PKA activities. This integrated two-component reporter system allow s one to focus specifically on particular signaling pathway endpoints and t he altered transactivation activity of either Elk1 or CREB. With the proced ures described here, many novel cell-based assays can be developed by gener ating new reporter cell lines with medically important but difficult-to-tra nsfect cell types, and by using different reporter genes or different fusio n transactivator genes.