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In Vivo Imaging of ß-Galactosidase Activity Using Far Red Fluorescent Switch

Center for Molecular Imaging Research, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts

ß-Galactosidase (ß-gal) has been widely used as a transgene reporter enzyme, and several substrates are available for its in vitro detection. The ability to image ß-gal expression in living animals would further extend the use of this reporter. Here we show that DDAOG, a conjugate of ß-galactoside and 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO), is not only a chromogenic ß-gal substrate but that the cleavage product has far-red fluorescence properties detectable by imaging. Importantly, the cleavage substrate shows a 50-nm red shift, enabling its specific detection in a background of intact probe, a highly desirable feature for in vivo imaging. Specifically, we show that ß-gal-expressing 9L gliomas are readily detectable by red fluorescence imaging in comparison with the native 9L gliomas. We furthermore show that herpes simplex virus amplicon-mediated LacZ gene transfer into tumors can be transiently and thus serially visualized over time. The results indicated that in vivo real-time detection of ß-gal activity is possible by fluorescence imaging technology.

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