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Engineering an effective Mn‐binding MRI reporter protein by subcellular targeting
Author(s) -
Bartelle Benjamin B.,
Mana Miyeko D.,
SueroAbreu Giselle A.,
Rodriguez Joe J.,
Turnbull Daniel H.
Publication year - 2015
Publication title -
magnetic resonance in medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.696
H-Index - 225
eISSN - 1522-2594
pISSN - 0740-3194
DOI - 10.1002/mrm.25566
Subject(s) - golgi apparatus , immunocytochemistry , microbiology and biotechnology , transfection , subcellular localization , endoplasmic reticulum , in vivo , hek 293 cells , reporter gene , in vitro , organelle , green fluorescent protein , transport protein , chemistry , biophysics , biology , biochemistry , cytoplasm , gene expression , gene , genetics , endocrinology
Purpose Manganese (Mn) is an effective contrast agent and biologically active metal, which has been widely used for Mn‐enhanced MRI (MEMRI). The purpose of this study was to develop and test a Mn binding protein for use as a genetic reporter for MEMRI. Methods The bacterial Mn‐binding protein, MntR was identified as a candidate reporter protein. MntR was engineered for expression in mammalian cells, and targeted to different subcellular organelles, including the Golgi Apparatus where cellular Mn is enriched. Transfected HEK293 cells and B16 melanoma cells were tested in vitro and in vivo, using immunocytochemistry, MR imaging and relaxometry. Results Subcellular targeting of MntR to the cytosol, endoplasmic reticulum and Golgi apparatus was verified with immunocytochemistry. After targeting to the Golgi, MntR expression produced robust R1 changes and T1 contrast in cells, in vitro and in vivo. Co‐expression with the divalent metal transporter DMT1, a previously described Mn‐based reporter, further enhanced contrast in B16 cells in culture, but in the in vivo B16 tumor model tested was not significantly better than MntR alone. Conclusion This second‐generation reporter system both expands the capabilities of genetically encoded reporters for imaging with MEMRI and provides important insights into the mechanisms of Mn biology which create endogenous MEMRI contrast. Magn Reson Med 74:1750–1757, 2015. © 2014 Wiley Periodicals, Inc.