Bartelle Benjamin B, Mana Miyeko D, Suero-Abreu Giselle A, Rodriguez Joe J, Turnbull Daniel H
Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, New York, USA.
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Magn Reson Med. 2015 Dec;74(6):1750-7. doi: 10.1002/mrm.25566. Epub 2014 Dec 17.
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.
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.
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.
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.
锰（Mn）是一种有效的造影剂和生物活性金属，已广泛用于锰增强磁共振成像（MEMRI）。本研究的目的是开发并测试一种用作MEMRI基因报告基因的锰结合蛋白。
细菌锰结合蛋白MntR被确定为候选报告蛋白。对MntR进行工程改造以在哺乳动物细胞中表达，并靶向不同的亚细胞器，包括细胞内锰富集的高尔基体。使用免疫细胞化学、磁共振成像和弛豫测量法对转染的HEK293细胞和B16黑色素瘤细胞进行体外和体内测试。
通过免疫细胞化学验证了MntR在细胞质、内质网和高尔基体中的亚细胞定位。靶向高尔基体后，MntR的表达在体外和体内的细胞中产生了显著的R1变化和T1对比度。与二价金属转运蛋白DMT1（一种先前描述的基于锰的报告基因）共表达，进一步增强了培养的B16细胞中的对比度，但在测试的体内B16肿瘤模型中，并不比单独的MntR显著更好。
这种第二代报告系统既扩展了用于MEMRI成像的基因编码报告基因的能力，又为产生内源性MEMRI对比度的锰生物学机制提供了重要见解。
