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Assess the In Vivo Activation of Signal Transduction Pathways with,,,PathDetect Reporting Systems

PathDetect cis-reporting systems contain tandem repeats of one of the following enhancer elements: cyclic AMP response element (CRE), serum response element (SRE), nuclear factor B (NF-kB), activator protein 1 (AP-1), serum response factor (SRF), or p53 binding sites. These enhancer elements, together with a TATA box, control the expression of the downstream luciferase reporter gene. When a cis-reporter vector and an experimental mammalian expression construct are cotransfected into mammalian cells, increased luciferase activity indicates transcription activation and the involvement of the gene product in signaling pathways converging at the cis-acting element. Because most enhancer elements are regulated by more than one transcription activator, each enhancer element of the cis-reporting systems can be used to monitor more than one signal transduction pathway. As a consequence, the PathDetect cis-reporting systems assess only the potential involvement of a gene product in a pathway. A drawback of cis-reporting systems is the higher background of luciferase expression, which is caused by the binding of endogenous transcription factors to the enhancer elements in the reporter vectors. For a more specific assessment, the PathDetect trans-reporting systems can be used.5

Specificity of Cis-Reporting Systems

Figure 4

Experiments demonstrating the activity of the cis-reporter plasmids are shown in Figure 4. For each experiment, 1 g of a cis-reporter vector was cotransfected into 1.5 x 105 HeLa cells together with an expression vector of a known activator, either MEK kinase (MEKK) or the catalytic subunit
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