Construction and Validation of a Novel Pathway Ratio Reporter System (PARRES)

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Medicine and Health Sciences


The Transforming Growth Factor-beta (TGFβ) superfamily is a large group of signaling molecules whose members play conserved roles in embryo specification, organogenesis, and tissue homeostasis. Structural considerations delineate the TGFβ superfamily into ligands that interact with ALK1/2/3/6 or ALK4/5/7, with the former being mostly BMPs and the latter being mostly Activins and TGFβs. Reports of distinctly opposing effects between the BMP/GDF and Activin/TGFβ pathways are becoming increasingly common. A major focus of our laboratory is elucidating the molecular mechanisms that allow the BMP/GDF and Activin/TGFβ pathways to antagonize one another. To accomplish this goal, we must create a first-of-its-kind biosensor reporter system (called the Pathway Ratio Reporter System, or PARRES) that will allow us to examine the real-time ratio of BMP/GDF vs. Activin/TGFβ in vivo. PARRES is a novel biosensor system that uses two independent fluorescent reporters that are linked through reciprocal RNA interference so that, through mutual antagonism, fluorescent signal occurs only when transcriptional activation through one pathway is dominant. Here, we focus on the crucial, initial characterization of PARRES – the maturation and half-life kinetics of the fluorescent proteins. Using HEK293 cells, we demonstrate that the maturation kinetic of the Emerald variant of Green Fluorescent Protein is acceptable but the half-life is far too long for our needs. We subsequently destabilize Emerald to an approximate half-life of 29 hours by appending a validated degradation domain. Current studies involve generating point mutations in the destabilized Emerald protein to further reduce its half-life. Once completed, this biosensor will be especially useful to our lab as a tool for examining the influence of canonical signaling by skeletal mesenchymal progenitors, a population known to rely on BMPs, GDFs, TGFβ and Activins to instruct their differentiation into tissue-specific cell types such as osteoblasts.


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