Vol. 3 No. 1 - Mar 2017

Tomography is a scientific journal for publication of articles in imaging research

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Ferritin–EGFP Chimera as an Endogenous Dual-Reporter for Both Fluorescence and Magnetic Resonance Imaging in Human Glioma U251 Cells Caihong Jiang 1 , Dongmei Wu 1 , and E. Mark Haacke 1,2 1 Shanghai Key Laboratory of Magnetic Resonance, East China Normal University, Shanghai, China and 2 Department of Radiology, Wayne State University, Detroit, Michigan Corresponding Author: E. Mark Haacke, PhD WSU MR Research Facility, HUH-MR Research/Radiology, 3990 John R Street, Detroit, Michigan 48201; E-mail: Key Words: ferritin, EGFP, dual-reporter human glioma U251 cells, tetracycline-regulated system, MRI Abbreviations: Enhanced green fluorescent protein (EGFP), magnetic resonance imaging (MRI), Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide dimethyl sulfoxide (MTT), ferric citrate ammonium (FAC), inductively coupled plasma mass spectroscopy (ICP-MS), tetracycline repressor protein (TetR), tetracycline (Tet) A unique hybrid protein ferritin– enhanced green fluorescent protein (EGFP) was built to serve as an endoge- nous dual reporter for both fluorescence and magnetic resonance imaging (MRI). It consists of a human ferri- tin heavy chain (an iron-storage protein) at the N terminus, a flexible polypeptide in the middle as a linker, and an EGFP at the C terminus. Through antibiotic screening, we established stable human glioma U251 cell strains that expressed ferritin–EGFP under the control of tetracycline. These cells emitted bright green fluores- cence and were easily detected by a fluorescent microscope. Ferritin–EGFP overexpression proved effective in triggering obvious intracellular iron accumulation as shown by Prussian blue staining and by MRI. Further, we found that ferritin–EGFP overexpression did not cause proliferation differences between experimental and control group cells when ferritin–EGFP was expressed for ,96 hours. Application of this novel ferritin–EGFP chimera has a promising future for combined optical and MRI approaches to study in vivo imaging at a cel- lular level. INTRODUCTION Glioma is among one of the most malignant tumors and is characterized by high levels of mortality and recurrence (1). Further, glioma cells show infiltrative growth and have no obvious boundaries with surrounding normal tissues. Precise noninvasive imaging is of great importance in tumor local- ization, metastasis detection, and subsequent therapy. Fluo- rescence imaging can provide noninvasive real-time dynamic observation of tumors. However, fluorescence has poor pene- tration capability, limiting its usage in deep-seated tumors. In contrast, magnetic resonance imaging (MRI) is not affected by the depth of target tissues and has high spatial resolution. However, the sensitivity of MRI is not high enough for performing cellular or molecular imaging. Therefore, to solve this problem, MRI contrast agents were invented using substances such as iron or gadolinium (2, 3). For example, iron oxide nanoparticles are often used in labeling cells (4-6). However, with the division of cells, the exoge- nous reporter is diluted, leading to a loss of signal change over time. Subsequently, several MRI reporter genes have been introduced into cells by either plasmids or slow virus transfection to serve as endogenous reporters (7, 8). Our focus will be on the use of ferritin as part of a reporter gene. Ferritin is an extensively studied iron-storage protein in the human body and plays an important role in maintaining the balance of iron metabolism (9, 10). Ferritin is composed of 2 types of subunits, both H and L subunits, namely, the heavy and the light chains. H subunits are the core subunits of iron storage in ferritin and can work as preferable endogenous MRI reporters (11, 12). In recent years, dual reporters that combine the advan- tages of fluorescence imaging and MRI have gradually become hotspots for noninvasive imaging studies. A dual reporter is usually composed of a fluorescent protein that is used for fluo- rescence imaging and a ferritin protein that is used for MRI (13-16). However, transgenic ferritin has usually been expressed separately from fluorescent protein and thus is not directly observed by fluorescence detection. There is also some contro- versy about the effects of ferritin overexpression on cells and in the body (11, 17-20). The reason behind these contradictory facts may be the various ferritin expression levels and different cell types that have been applied in those studies. Here, we propose an improved dual-reporter ferritin– en- hanced green fluorescent protein (EGFP) chimera, with a human ADVANCES IN BRIEF ABSTRACT © 2017 The Authors. Published by Grapho Publications, LLC This is an open access article under the CC BY-NC-ND license ( ISSN 2379-1381 TOMOGRAPHY.ORG | VOLUME 3 NUMBER 1 | MARCH 2017 1

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