Abstract
We are developing methods to image molecular and cellular events in living subjects. In this study, we validate imaging of protein-protein interactions in living mice by using bioluminescent optical imaging. We use the well studied yeast two-hybrid system adapted for mammalian cells and modify it to be inducible. We employ the NF-kappaB promoter to drive expression of two fusion proteins (VP16-MyoD and GAL4-ID). We modulate the NF-kappaB promoter through tumor necrosis factor alpha. Firefly luciferase reporter gene expression is driven by the interaction of MyoD and ID through a transcriptional activation strategy. We demonstrate the ability to detect this induced protein-protein interaction in cell culture and image this induced interaction in living mice by using transiently transfected cells. The current approach will be a valuable and potentially generalizable tool to noninvasively and quantitatively image protein-protein interactions in living subjects. The approaches validated should have important implications for the study of protein-protein interactions in cells maintained in their natural in vivo environment as well as for the in vivo evaluation of new pharmaceuticals targeted to modulate protein-protein interactions.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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Cell Culture Techniques
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Cell Line, Transformed
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism*
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Fungal Proteins / genetics
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Fungal Proteins / metabolism
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Gene Expression
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Genes, Reporter
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Herpes Simplex Virus Protein Vmw65 / genetics
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Herpes Simplex Virus Protein Vmw65 / metabolism
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Humans
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Inhibitor of Differentiation Protein 1
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Luciferases / genetics
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Mice
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MyoD Protein / genetics
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MyoD Protein / metabolism*
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NF-kappa B / genetics
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Promoter Regions, Genetic
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Proteins / metabolism*
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Rats
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Repressor Proteins*
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Saccharomyces cerevisiae Proteins*
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Transcription Factors / genetics
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Transcription Factors / metabolism*
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Tumor Cells, Cultured
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Tumor Necrosis Factor-alpha / pharmacology
Substances
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DNA-Binding Proteins
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Fungal Proteins
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GAL4 protein, S cerevisiae
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Herpes Simplex Virus Protein Vmw65
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ID1 protein, human
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ID1 protein, rat
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Idb1 protein, mouse
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Inhibitor of Differentiation Protein 1
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MyoD Protein
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NF-kappa B
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Proteins
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Recombinant Fusion Proteins
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Repressor Proteins
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Saccharomyces cerevisiae Proteins
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Transcription Factors
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Tumor Necrosis Factor-alpha
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Luciferases