450 VOLUME 11 | NUMBER 4 | APRIL 2005 NATURE MEDICINE A new transgene reporter for in vivo magnetic resonance imaging Guillem Genove 1 , Ulrike DeMarco 1 , Hongyan Xu 1 , William F Goins 2 & Eric T Ahrens 1 We report a new platform technology for visualizing transgene expression in living subjects using magnetic resonance imaging (MRI). Using a vector, we introduced an MRI reporter, a metalloprotein from the ferritin family, into specific host tissues. The reporter is made superparamagnetic as the cell sequesters endogenous iron from the organism. In this new approach, the cells construct the MRI contrast agent in situ using genetic instructions introduced by the vector. No exogenous metal-complexed contrast agent is required, thereby simplifying intracellular delivery. We used a replication- defective adenovirus vector to deliver the ferritin transgenes. Following focal inoculation of the vector into the mouse brain, we monitored the reporter activity using in vivo time-lapse MRI. We observed robust contrast in virus-transduced neurons and glia for several weeks. This technology is adaptable to monitor transgene expression in vivo in many tissue types and has numerous biomedical applications, such as visualizing preclinical therapeutic gene delivery. The ability to image the duration and location of gene expression in vivo and noninvasively is important for the future of biology and clinical medicine. The emerging field of genetic medicine requires in vivo imaging methods that can indicate where and when therapeutic gene delivery has occurred. Gene expression is commonly imaged using an optically visible reporter that is coexpressed with the gene of interest. Reporter detection often involves killing the animal for histochemical detection in fixed tissues. In vivo detection of gene expression using biophotonic reporters 1 or positron emission tomog- raphy reporters 2 shows great promise for many applications; however, overcoming tissue opacity and/or resolution limitations still remains a key challenge. MRI is a widely used clinical diagnostic tool because it is noninvasive, allows views into opaque subjects and provides soft-tissue contrast at reasonably high spatial resolution. Several pioneering papers have put forth the notion of using metal-complexed MRI agents to dis- play transgene activity in vivo 3,4 , however, these approaches have not yet achieved widespread utility. Delivering the metal complexes is a challenge because they generally have poor penetrance into tissues and cells. Kinase transgenes have also been investigated as reporters using in vivo nuclear magnetic resonance (NMR) 5,6 . Here we present a new approach that uses MRI to visualize transgene expression in vivo through a gene-transfer vector encoding a marker gene. This approach yields robust image contrast and is widely applicable to mammalian systems. The basis of our approach is as follows. In nature, many organisms have evolved to produce intracellular, biomineralized, superparamagnetic nanocrystals. The magnetosome structure in magnetotactic bacteria is a dramatic example 7 . Other examples include certain metalloproteins, such as ferritin. Ferritin is ubiquitous and highly conserved throughout almost all organisms. Because of the crystalline ferrihydrite core, ferritin has an anomalously high superparamagnetism 8 and a marked effect on solvent NMR relaxation rates 8–11 . Thus, ferritin is an ideal molecule to express in in vivo MRI studies. In this study we introduced an MRI reporter gene encoding metalloproteins from the ferritin family into specific tissues of a living subject using a replication-defective adenovirus (AdV). The vector-encoded reporter is made super- paramagnetic as the cell sequesters endogenous iron (Fe) from the organism. Our approach is unique because the cell constructs the MRI contrast agent in situ using vector-mediated instructions. No bulky metal complex is required, thereby simplifying intracellular delivery. We initially characterized the biological effects of reporter expression in a series of in vitro assays. For in vivo experiments, we delivered the AdV-ferritin vector (AdV-FT) into the brain of C57Bl/6J mice by stereotaxic injection. The virus-transduced neurons and glia showed robust image contrast. We longitudinally monitored the contrast enhancement for up to 5 weeks, the endpoint of our study. A LacZ-expressing AdV control vector (AdV-LacZ) injected into the contralateral side showed no MRI contrast, but histology showed a similar transduction pattern as the AdV-FT vector. Moreover, immunohistochemical detection of the ferritin transgenes corroborated the pattern shown by MRI. Overall, this technology can be adapted to examine gene expression in many tissue types, therefore an immense number of preclinical in vivo applications exist. Examples include preclinical testing of gene therapeutics to assess dosage protocols and visualizing gene expression in transgenic animals. 1 Department of Biological Sciences and Pittsburgh NMR Center for Biomedical Research, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, USA. 2 Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, 200 Lothrop Street, Pittsburgh, Pennsylvania, 15213, USA. Correspondence should be addressed to E.T.A. (eta@andrew.cmu.edu). Published online 20 March 2005; doi:10.1038/nm1208 TECHNICAL REPORTS © 2005 Nature Publishing Group http://www.nature.com/naturemedicine