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PLoS By Category | Recent PLoS Articles
Oncology - Radiology and Medical Imaging

Near-Infrared Fluorescence Imaging of Mammalian Cells and Xenograft Tumors with SNAP-Tag
Published: Friday, March 30, 2012
Author: Haibiao Gong et al.

by Haibiao Gong, Joy L. Kovar, Brenda Baker, Aihua Zhang, Lael Cheung, Daniel R. Draney, Ivan R. Corrêa, Ming-Qun Xu, D. Michael Olive

Fluorescence in the near-infrared (NIR) spectral region is suitable for in vivo imaging due to its reduced background and high penetration capability compared to visible fluorescence. SNAPf is a fast-labeling variant of SNAP-tag that reacts with a fluorescent dye-conjugated benzylguanine (BG) substrate, leading to covalent attachment of the fluorescent dye to the SNAPf. This property makes SNAPf a valuable tool for fluorescence imaging. The NIR fluorescent substrate BG-800, a conjugate between BG and IRDye 800CW, was synthesized and characterized in this study. HEK293, MDA-MB-231 and SK-OV-3 cells stably expressing SNAPf-Beta-2 adrenergic receptor (SNAPf-ADRß2) fusion protein were created. The ADRß2 portion of the protein directs the localization of the protein to the cell membrane. The expression of SNAPf-ADRß2 in the stable cell lines was confirmed by the reaction between BG-800 substrate and cell lysates. Microscopic examination confirmed that SNAPf-ADRß2 was localized on the cell membrane. The signal intensity of the labeled cells was dependent on the BG-800 concentration. In vivo imaging study showed that BG-800 could be used to visualize xenograph tumors expressing SNAPf-ADRß2. However, the background signal was relatively high, which may be a reflection of non-specific accumulation of BG-800 in the skin. To address the background issue, quenched substrates that only fluoresce upon reaction with SNAP-tag were synthesized and characterized. Although the fluorescence was successfully quenched, in vivo imaging with the quenched substrate CBG-800-PEG-QC1 failed to visualize the SNAPf-ADRß2 expressing tumor, possibly due to the reduced reaction rate. Further improvement is needed to apply this system for in vivo imaging.
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