Screening of plant factors that modulate the activation of the NF-kB transcription factor in mammalian cells
Önal, Pınar (2007) Screening of plant factors that modulate the activation of the NF-kB transcription factor in mammalian cells. [Thesis]
The signal transduction pathway that leads to the activation of NF-kB is extensively studied because of its importance in immune system activation. Any molecule that interferes with the pathway is therapeutically important because of its potential role in triggering or inhibiting the immune response. To identify proteins that interfere with the activation of NF-kB, we designed and performed a genetic screening system of plant proteins. Our screen consisted of ankyrin repeat plant proteins expressed in mammalian tissue culture cells along with an NF-kB reporter vector that expressed Green Fluorescent Protein (GFP) upon NF-kB activation. As a tissue culture system we used the easily transfected human embryonic kidney cell line FEK293T. To perform our screen, we generated an NF-kB sensitive GFP reporter plasmid by standard molecular biology cloning techniques and we constructed a library of mammalian expression vectors that contained 90 A. thaliana cDNAs by Cre-mediated recombination between plasmids. We restricted our analysis to 90 cDNAs of ankyrin repeat domain containing proteins from A. thaliana for three reasons: First, ankyrin repeat containing proteins encoded by the mammalian genome have been shown to directly bind to NF-kB, second, proteins encoded by different genomes have the potential to interact with higher affinity than those encoded by the same genome and finally, some A. thaliana ankyrin repeat domain proteins have been shown to be involved in plant immune responses. In our screen we found out that three out of 90 plant cDNAs that were expressed in mammalian cells affected TNF-ot induced NF-kB activation by changing GFP expression from our reporter vector.
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