Self-help for in vivo Imaging

CONTRAST AND COMPARE:

Courtesy of Nicole Reading and Eric Ahrens, Carnegie Mellon UniversityThe gene for an iron-storage protein (green) is incorporated into an adenovirus gene-delivery vehicle, or vector, and delivered to the brain via injection. The metal-binding protein acts like a nanoscale magnet and a potent MRI "reporter." When the subject is placed in an MRI magnet, the brain cells that contain the reporter can be identified and imaged at high resolution (arrow). The MRI reporter gene can be linked to any other gene of interest (e.g., a therapeutic gene).Visualizing gene expression in living animals, at high spatial resolution and in real time, has been a long-held goal in molecular research. Now, a new platform technology, based on a well-known tool with a clever twist, is enabling molecular biologists to do just that.The new technology uses magnetic resonance imaging (MRI) to detect the location and duration of transgene expression in vivo, without the burden of having to externally introduce an MRI contrast agent. In fact, the contrast agent is produced by the transgenic cells themselves.3"Usually, MRI agents are exogenous compounds that are cooked in a flask and have to be delivered to the cells of interest at a reasonable concentration. That's a challenge," says coauthor Eric T. Ahrens of Carnegie Mellon University in Pittsburgh, Pa. Researchers have already used MRI successfully to monitor gene expression,12 but delivering the often bulky metal-complexed agents can be a limitation."The delivery is particularly problematic in tissues such as brain, because the contrast agents cannot pass the blood-brain barrier," says William F. Goins, a coauthor from the University of Pittsburgh. In addition, when coupled with a viral gene-delivery vector, some cells take up the virus and express the gene product, while others take up the contrast product, but not all cells take up both.To overcome these problems, Ahrens and Goins teamed up with three colleagues and constructed a replication-defective adenovirus vector that contained a gene for an MRI reporter – a member of the ferritin family that occurs naturally in cells and sequesters iron molecules from its surroundings.They then introduced the vector into the brains of living mice and imaged the reporter expression for more than a month. The rationale behind this approach was that when the overexpressed ferritin sequestered iron into its core, it would enhance its own contrasting effect, which it did. The study rendered robust and sharp images of transduced cells, and showed no overt toxicity in the mouse brain from the MRI reporter."People need a benign way to follow the location of vectors and transgene expression," says Joseph C. Glorioso of the University of Pittsburgh, who was not involved in the study. "Most techniques used at present require killing the animal and doing sections. Here you have real-time expression in intact subjects. Moreover, the reporter they used is a natural protein, so you don't have to worry too much about immune-related toxicity. It's a great way to do it."Michal Neeman's laboratory at the Weizmann Institute of Science in Israel has also used ferritin as an endogenous reporter, in this case, to detect transgene expression in C6 glioma tumors.4 "Their approach was different from ours," says Ahrens. "They generated a stable cell-line expressing ferritin and then injected it into animals to see whether they could monitor the tumor cells as they divided and grew. It's an excellent paper."The Neeman's group's ferritin reporter includes features that enable cross-validation while improving specificity. "This is particularly important for MRI, because signal changes can reflect pathological changes such as bleeding or scarring. Contrast changes alone are not sufficient for accurately assigning them to the transgene reporter," says Neeman.Neeman's reporter carries a specific tag in the transgenic ferritin's code that enables its separation from the endogenous natural ferritin, and a second reporter that enables independent detection by optical imaging. In addition, the entire MRI reporter is placed under a switchable, tetracycline-sensitive gene-expression system. "This means that only contrast changes induced by the antibiotic are due to the expression of the reporter."Neeman's laboratory is interested in questions about tissue remodeling, and will use this technology to study changes in gene expression during the process of angiogenesis. Ahrens and his colleagues intend to monitor therapeutic gene expression in vivo in animal disease models, to obtain preclinical data necessary to initiate human clinical trials.

Self-help for in vivo Imaging

Visualizing gene expression in living animals, at high spatial resolution and in real time, has been a long-held goal in molecular research.

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