In another example, we investigated the effects of varying the amounts of both the reporter vector and the expression vector using the PathDetect SRE cis-reporting system (Figure 2, panel B). These results showed that, within a certain range, when the pSRE-Luc reporter plasmid increased, more pFC-MEKK expression vector (x-axis) was required to achieve maximum activation of luciferase expression. Since the ratio of reporter plasmid to expression vector affects activation, optimizing experimental conditions should include optimizing the amounts of both plasmids.
Both the PathDetect in vivo trans- and cis- reporting systems are unique tools for studying signal transduction pathways and the functions of new genes. However, as we demonstrated in this report, plasmid amounts used for cotransfection dramatically effect luciferase expression. Therefore, to gain the best results, researchers should first implement a study to optimize these conditions.
Transfections were performed using a standard lipofection method. Each
experiment was performed in duplicate and shows the average value for both
experiments. As the different transfected plasmids varied, the total DNA
amount for each transfection was kept constant with different amounts of an
unrelated plasmid (pBluescript plasmid).
After transfecting, cells were maintained in Dulbeccos minimal essential
medium containing 0.5% fetal bovine serum. The pFR-Luc reporter plasmid, which
contains the entire coding sequence of the luciferase gene downstream from a
basic promoter element (TATA box) joined to five tandem repeats of the 17-bp
GAL4 binding element, was used i