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Optimizing Transfection Conditions for Studying Signal Transduction Pathways

ion trans-activator plasmids, a reporter plasmid that encodes the firefly luciferase gene, and positive control plasmids.1 The fusion trans-activator plasmids express pathway-specific trans-activator proteins that consist of the yeast GAL4 DNA binding domain and the activation domains of various transcription factors. When a fusion trans-activator plasmid, reporter plasmid, and uncharacterized gene are cotransfected into mammalian cells, the uncharacterized gene product can directly or indirectly phosphorylate the fusion trans-activator protein and activate transcription of the luciferase gene from the reporter plasmid. Luciferase activity that exceeds background levels indicates that the gene of interest is involved in the pathway being evaluated.

Enhancer elements are the convergent points for many intracellular signal transduction pathways. The PathDetect cis-reporting systems were designed to quickly and easily assess the in vivo activation of these pathways. The PathDetect cis-reporting systems8 encompass a reporter plasmid that contains the luciferase reporter gene driven by a basic promoter element (TATA box) joined to tandem repeats of either AP-1, CRE, SRE, NF-kB, p53, or SRF binding elements. When a plasmid that expresses the gene of interest is cotransfected into mammalian cells with a cis-reporter plasmid, increased luciferase expression indicates either direct or indirect transcriptional activation. The PathDetect cis-reporting systems can also be used to evaluate the effects of extracellular stimuli or compounds of interest on each particular enhancer element.

Cell lines possess unique properties, such as the amount and activity of endogenous protein kinases and the repertoires of transcription factors, that are determined by their origins and passages. In addition, different laboratories and researchers adopt somewhat different protocols
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