Obtaining the best results with PathDetect in vivo reporting systems
Tim Sanchez Li Xu Mary Buchanan Chao-Feng Zheng
Stratagenes PathDetect in vivo signal transduction pathway reporting systems are used to study activation of specific signaling pathways by uncharacterized gene products, extracellular stimuli, or drug candidates. Many different cell lines may be used, and each one used in transfections has a unique genetic and biochemical background. As a result, the expression of gene products and, hence, the optimal amount of plasmid needed for transfections, varies in different cell lines. To achieve the best results with PathDetect systems, it may be necessary to first optimize transfections with the desired system. In this article, we report the results of several experiments to show that the readout signals of the PathDetect trans- and cis-reporting systems are dramatically influenced by the amounts of DNA used for transfections.
The PathDetect in vivo signal transduction pathway trans-reporting systems1 are used to study the in vivo effects of new genes, growth factors, drug candidates, and extracellular stimuli on the activation of c-Jun N-terminal kinase2,3 (JNK), mitogen-activated protein kinase4,5 (MAPK), cyclic AMP-dependent protein kinase6 (PKA), and other signaling molecules that lead to the activation of these kinases. Stratagene has continued to update the PathDetect trans-systems to study signal transduction events that converge at the transcription factors CHOP (activated by p38 MAPK), ATF2 and c-Fos.6,7
The PathDetect trans-reporting systems include unique fus 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 for culturing and transfecting cell lines. Because of these variables, it is necessary to optimize experimental conditions to achieve the best results that the PathDetect reporting systems offer. Although many experimental conditions affect the results of PathDetect systems, the amounts of the fusion activator plasmid and expression vector for the gene of interest most dramatically affect the trans-reporting systems. When using the cis-reporting systems, amounts of the reporter plasmid or expression vector can be easily optimized for superior results.
By varying the amounts of the pFA-ATF2 plasmid, we optimized the conditions for transient transfections of HeLa cells (Figure 1, panel A). The GAL4-ATF2 fusion protein, which is expressed from the pFA-ATF2 plasmid, is strongly activated by the MEKK protein in transient transfection assays.9,10 In this experiment, HeLa cells (1.5 x 105/well) were cotransfected with the indicated amounts of the pFA-ATF2 plasmid, 0.5 g of the pFR-Luc plasmid, and 25 ng of the pFC-MEKK plasmid. The strongest signal for luciferase activity was seen for transfections when 10 ng of the pFA-ATF2 plasmid was used. As the amount of the pFA-ATF2 plasmid further increased, luciferase expression decreased.
When transcription factors are overexpressed, some components of the transcription machinery become depleted; this is described as squelching, and results in decreased transcription.11 One possible explanation for decreased luciferase expression was that the concentration of the GAL4-ATF2 fusion protein e xceeded the level able to be phosphorylated by a limited amount of upstream activators in the cells. Phosphorylated and nonphosphorylated species of the GAL4-ATF2 fusion compete for the GAL4 binding sites on the reporter plasmid. Because transcription of the luciferase gene on the reporter plasmid is activated only by the phosphorylated GAL4-ATF2 fusion protein, lower levels of luciferase activity may occur.
As a negative control, we varied the amounts of the pFA2-dbd plasmidwhich does not contain an activation sequencein parallel transfections in place of the pFA-ATF2 trans-activator plasmid. As expected, this control showed very low background luciferase expression.
Transfecting varying amounts of the expression plasmid containing the gene of interest also had dramatic effects. Hence, another way to optimize induction of the PathDetect system is by transfecting varying amounts of the expression plasmid while maintaining constant amounts of the trans-activator plasmid and reporter plasmid.
In Figure 1, panel B, HeLa cells (1.5 x 105/well) were cotransfected with 10 ng of the pFA-ATF2 plasmid, 0.5 g of the pFR-Luc plasmid, and varying amounts of the pFC-MEKK plasmid. Maximum activation of luciferase expression was seen when 10 ng of the pFC-MEKK plasmid was used per transfection. In this experiment, when the amount of the pFC-MEKK plasmid increased above 10 ng, luciferase activity declined. As before, the GAL4-ATF2 fusion protein is phosphorylated by overexpressing the MEKK protein and binds to the GAL4 binding domain. However, in this scenario, too much of the MEKK protein could cause other signal transduction pathways to overactivate, which would deplete some of the common factors (e.g., coactivators) in the cells transcription machinery. When factors are depleted, they result in a decreased overall transcription rate of the cell, including transcription of the luciferase gene in the reporter plasmid.
In this experiment, we employed two different negative controls: the pFA2-dbd plasmid was used in place of the pFA-ATF2 plasmid and the pcmv-script expression vector without an insert was used in place of the pFC-MEKK plasmid.
Since endogenous transcription factors interact with the enhancer elements used in the PathDetect cis-reporting systems, a higher background often results. Optimizing conditions for the experimental and control plasmids or stumuli minimizes such problems. To demonstrate how the cis-reporting systems can be optimized in this situation, we cotransfected the pAP1-Luc reporter vector with either the pFC-MEKK expression plasmid or the empty vector without insert, the pCMV-Script plasmid, into HeLa cells. While holding the concentration of the pAP1-Luc plasmid constant, we varied the amount of the pFC-MEKK plasmid (Figure 2, panel A). Optimized activation of the pAP1-Luc reporting vectors was seen when 10 ng of pFC-MEKK was used.
Sufficient plasmid needs to be available to produce protein to activate signaling pathways that lead to luciferase expression; however, as with the trans-reporting systems, when the transcription activator is too powerful, it can squelch the signal by titrating cellular components necessary for the cellular transcription machinery and other processes. When more than 10 ng of the pFC-MEKK plasmid was used, the system was probably squelched, and activation decreased. The pCM V-Script plasmid, the negative control for the pFC-MEKK plasmid, did not stimulate luciferase expression from the reporter vector.
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 n the experiment to optimizing the trans-reporting system. The cis-reporter plasmid was used in experiments to optimize the cis-reporting system, as indicated in the Figure legends. Cell lysates were prepared, and luciferase assays were performed according to the manual for Stratagenes Luciferase Assay Kit.
Xu, L., Sanchez, T., and Zheng, C.-F. (1997) Strategies 10: 1-3.
Lin, A., et al. (1995) Science 268: 286-289.
Minden, A., et al. (1994) Science 266: 1719-1723.
Price, M.A., et al. (1995) EMBO J. 14: 2589-2601.
Marais, R., Wynne, J., and Treisman, R. (1993) Cell 73: 381-393.
Smeal, T., Hibi, M., and Karin, M. (1994) EMBO J. 13: 6006-6010.
Xu, L., Sanchez, T., and Zheng, C.-F. (1997) Strategies 10: 79-80.
Xu, L., Sanchez, T., Buchanan, M., and Zheng, C.-F. (1997) Strategies 10: 121-123.
Livingstone, C., Patel, G. and Jones, N. (1995) EMBO J. 14: 1785-1797.
Maekawa, T., et al. (1989) EMBO J. 8: 2023-2028.
Gill, G. and Ptashne, M. (1988) Nature 334: 721-724.