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

protein12 the pFC-MEK1 plasmid, constitutively expressing this kinase, was used to demonstrate the specificity of the pFA2-Elk1 plasmid. Cotransfection of the pFA2-Elk1 plasmid with the pFR-Luc plasmid and pFC-MEK1 plasmid showed a dramatic increase in luciferase activity. The parallel, control cotransfection of the pFC2-dbd, pFR-Luc, and pFC-MEK1 plasmids showed negligible luciferase activity. Similar experiments have also been performed for the pFA-ATF-cFos and pFA-CHOP plasmids (data not shown).

Cis-Reporting Systems: Looking for Initial Clues

Cis-acting enhancer elements usually bind more than one transcriptional factor and, hence, can interact with a wide spectrum of signals. For this reason, Stratagene designed the PathDetect cis-reporting systems as another approach to study signal transduction pathways. By using these systems, it is possible to assess the effects of a gene product or extracellular stimulus with specific enhancer elements. Binding of an activated transcription factor to specific enhancer elements, which are found in the promoters of various cellular genes, is often the final step in many activational series. The activation of involved signaling events is reflected by the transcriptional level of the cellular genes. For Stratagenes cis-reporting systems, activation of the luciferase reporter gene, rather than a cellular gene, is used to indicate activation status.

Figure 3

Each of the PathDetect in vivo signal transduction pathway cis-reporting systems includes one of six cis-reporter vectors and a positive control plasmid. Positive control plasmids express gene products known to activate the signaling pathway converging at the corresponding enhancer elements. The six reporter plasmids (Figure 3) of the
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