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Improved Vectors for PathDetect Trans-Reporting Systems

In vivo readout of signal transduction pathway activation

Li Xu Tim Sanchez Mary Buchanan Chao-Feng Zheng
Stratagene

Stratagenes PathDetect in vivo signal transduction pathway reporting systems can be used to assess the in vivo activation of signal transduction pathways. To expand the utility of these systems, Stratagene is replacing the pFA-cJun, pFA-CREB and pFA-Elk1 plasmids with new, improved fusion trans-activator plasmids. The pFA2-cJun, pFA2-CREB and pFA2-Elk1 plasmids feature the cytomegalovirus (CMV) promoter for expression of fusion proteins in a wide variety of cell lines and the neomycin-resistance gene for facilitating selection of stable cell lines.

Stratagene recently introduced the PathDetect in vivo signal transduction pathway trans-reporting systems, which are designed for specific, rapid and convenient assessment of the in vivo activation of signal transduction pathways.1 Researchers are successfully using these reporting systems for studying 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 kinase6,7 (PKA) and other signaling molecules leading to the activation of these kinases. Each PathDetect reporting system includes (1) a pathway-specific fusion trans-activator plasmid, which is an in-frame fusion of an activation domain and the DNA binding domain of the yeast trans-activator GAL4,6,8 (2) the pFR-Luc reporter plasmid for expression of the Photinus pyralis (firefly) luciferase gene controlled by a synthetic promoter that contains the yeast GAL4 upstream activating sequences (UAS) (3) a positive control plasmid that is known to activate the trans-activator protein and (4) a negative control plasmid that contains only the DNA binding domain of the yeast GAL4. When a fusion trans-activator plasmid, reporter plasmid and gene of interest are cotransfected into mammalian cells, either direct or indirect phosphorylation of the fusion trans-activator protein by the uncharacterized gene product will cause transcription of the luciferase gene from the reporter plasmid. Luciferase activity, which can be conveniently assayed using Stratagenes Luciferase Assay Kit, is an indication that the gene of interest is involved in the pathway being evaluated.

Recently, Stratagene expanded the PathDetect reporting systems by introducing the pFA-ATF2 and pFA-cFos fusion trans-activator plasmids8 for studying signal transduction pathways that converge at the transcription factors ATF2 and c-Fos, respectively. At the same time, Stratagene introduced the pFA-CMV vector.9 This special vector can be used for easy cloning of the activation domain of any transcription factor sequence and subsequent expression of GAL4 fusion proteins in a variety of mammalian cells. The pFA-CMV vector features a multiple cloning site with 10 unique, conveniently arranged restriction sites for insertion of any activation domain sequence; the CMV promoter for high-level expression of fusion trans-activator proteins in many mammalian cell lines and the neomycin-resistance gene, which facilitates selection of stable cell lines that express fusion trans-activator proteins.

Improved Fusion Trans-Activator Plasmids

To upgrade the PathDetect reporting systems, Stratagene is replacing the pFA-cJun, pFA-CREB and pFA-Elk1 plasmids with improved fusion trans-activator plasmids. The new pFA2-cJun, pFA2-CREB and pFA2-Elk1 plasmids were derived from the pFA-CMV vector and offer expression driven by the powerful CMV promoter and the capability for G418 selection of stable cell lines. A new negative control plasmid, the pFC2-dbd plasmid, completes this se t of improved vectors. This plasmid expresses the GAL4 DNA binding domain without a transcriptional activator and has the same backbone as the other members of the pFA2 vector series. All plasmids of the PathDetect reporting systems are highly purified and extensively tested, and plasmid sequences are available upon request.

Activity of the pFA2-cJun, pFA2-CREB and pFA2-Elk1 Plasmids

Expression of GAL4 fusion proteins in HeLa cells was confirmed by Western blot analysis using a monoclonal antibody that recognizes the GAL4 DNA binding domain (data not shown). The pFA2-cJun, pFA2-CREB and pFA2-Elk1 plasmids were also tested in transient transfection assays in HeLa cells. For each transfection, the activity and specificity of the new fusion trans-activator plasmids were compared to the original plasmids. As a negative control for these experiments, we included a set of transfections with the pBK-CMV plasmid, a construct that does not contain the GAL4 DNA binding domain sequence and does not express protein from the CMV promoter.

The pFR-Luc plasmid (0.5 g) and pFC-MEKK (25 ng) plasmid were cotransfected into HeLa cells together with different amounts of either the pFA-cJun or pFA2-cJun plasmids (figure 1, panel A). The pFC-dbd plasmid was used as the negative control for the pFA-cJun plasmid; the pFC2-dbd plasmid was used as the negative control for the pFA2-cJun plasmid. The transcription activating levels for both control pFC-dbd and pFC2-dbd plasmids were very low, yielding negligible luciferase activity. Optimized luciferase expression was seen when only 1 ng of the pFA2-cJun plasmid was used, while optimized expression for the pFA-cJun plasmid required 10 ng. This observed difference is presumed to result from the strength of the CMV promoter.

The activation and specific ity of the pFA-CREB and pFA2-CREB plasmids were compared using the PKA protein, a known activator of the CREB protein. When either of the trans-activator plasmids was cotransfected with the pFR-Luc plasmid and the pFC-PKA plasmid, comparable levels of luciferase activity were measured (figure 1, panel B). Cotransfection of the pFR-Luc and pFC-PKA plasmids with either the pFC-dbd or pFC2-dbd plasmids yielded similar background levels of luciferase activity.

Since the MEK1 protein is a known upstream activator of the Elk1 protein, the pFC-MEK1 plasmid was used to compare the activity of the pFA-Elk1 and pFA2-Elk1 plasmids. The background luciferase activity for this experiment, as measured by cotransfection of the pFR-Luc plasmid with the pFC2-dbd plasmid and the pFC-MEK1 plasmid, was lower than the background for the parallel control experiment that used the pFC-dbd plasmid. When these differences in background activity were normalized, cotransfection of either the pFA-Elk1 plasmid or pFA2-Elk1 plasmid with the pFC-MEK1 and pFR-Luc plasmids yielded comparable luciferase activity (figure 1, panel C).

Conclusions

Stratagene is upgrading the successful PathDetect reporting systems by offering new and improved fusion trans-activator plasmids. As measured in transient transfection assays, the new pFA2-cJun, pFA2-CREB and pFA2-Elk1 plasmids offer activity and specificity that are comparable to the original trans-activator plasmids. However, as a result of expression driven from the strong CMV promoter, these new plasmids can be used in a wide variety of cell lines. In addition, the neomycin-resistance gene of the pFA2-cJun, pFA2-CREB and pFA2-Elk1 plasmids facilitates stable selection of cell lines that express the fusion trans-activator protein.

REFERENCES
  1. Xu, L., Sanchez, T., and Zheng, C.-F. (1997) Strategies 10: 1-3.

  2. Lin, A., et al. (1995) Science 268: 286-289.

  3. Minden, A., et al. (1994) Science 266: 1719-1723.

  4. Price, M.A., et al. (1995) EMBO J. 14: 2589-2601.

  5. Marais, R., Wynne, J., and Treisman, R. (1993) Cell 73: 381-393.

  6. Smeal, T., Hibi, M., and Karin, M. (1994) EMBO J. 13: 6006-6010.

  7. Xing, J., et al. (1996) Science 273: 959-963.

  8. Sadowski, I., and Ptashne, M. (1989) Nucleic Acids Res. 17: 7539.

  9. Xu, L., and Zheng, C.-F. (1997) Strategies 10: 81-83.


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