( Optimizing Transfection Conditi...)Optimizing Transfection Conditions for Studying Signal Transduction Pathways

Obtaining the best results with PathDetect in vivo reporting systems

Tim Sanchez Li Xu Mary Buchanan Chao-Feng Zheng
Stratagene

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 kinase^2,3 (JNK), mitogen-activated protein kinase^4,5 (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
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