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Near-infrared fluorescence imaging of mammalian cells and xenograft tumors with SNAP-tag.

ABSTRACT: 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. SNAP(f) 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 SNAP(f). This property makes SNAP(f) 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 SNAP(f)-Beta-2 adrenergic receptor (SNAP(f)-ADR?2) fusion protein were created. The ADR?2 portion of the protein directs the localization of the protein to the cell membrane. The expression of SNAP(f)-ADR?2 in the stable cell lines was confirmed by the reaction between BG-800 substrate and cell lysates. Microscopic examination confirmed that SNAP(f)-ADR?2 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 SNAP(f)-ADR?2. 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 SNAP(f)-ADR?2 expressing tumor, possibly due to the reduced reaction rate. Further improvement is needed to apply this system for in vivo imaging.

SUBMITTER: Gong H

PROVIDER: S-EPMC3316518 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Near-infrared fluorescence imaging of mammalian cells and xenograft tumors with SNAP-tag.

Gong Haibiao H Kovar Joy L JL Baker Brenda B Zhang Aihua A Cheung Lael L Draney Daniel R DR Corrêa Ivan R IR Xu Ming-Qun MQ Olive D Michael DM

PloS one 20120330 3

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. SNAP(f) 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 SNAP(f). This property makes SNAP(f) a valuable tool for fluorescence imaging. The NIR fluorescent substrate BG-800, a conjugate bet ...[more]

PMID: 22479502

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Project description:We have developed a novel reporter assay that leverages SNAP-epitope tag/near-infrared (NIR) imaging technology to monitor G protein-coupled receptor (GPCR) degradation in human cell lines. N-terminal SNAP-tagged GPCRs were subcloned and expressed in human embryonic kidney (HEK) 293 cells and then subjected to 24 h of cycloheximide (CHX)-chase degradation assays to quantify receptor degradation half-lives (t1/2) using LICOR NIR imaging-polyacrylamide gel electrophoresis (PAGE) analysis. Thus far, we have used this method to quantify t1/2 for all nine adrenergic (ADRA1A, ADRA1B, ADRA1D, ADRA2A, ADRA2B, ADRA2C, ADRB1, ADRB2, ADRB3), five somatostatin (SSTR1, SSTR2, SSTR3, SSTR4, SSTR5), four chemokine (CXCR1, CXCR2, CXCR3, CXCR5), and three 5-HT2 (5HT2A, 5HT2B, 5HT2C) receptor subtypes. SNAP-GPCR-CHX degradation t1/2 values ranged from 0.52 h (ADRA1D) to 5.5 h (SSTR3). On the contrary, both the SNAP-tag alone and SNAP-tagged and endogenous β-actin were resistant to degradation with CHX treatment. Treatment with the proteasome inhibitor bortezomib produced significant but variable increases in SNAP-GPCR protein expression levels, indicating that SNAP-GPCR degradation primarily occurs through the proteasome. Remarkably, endogenous β2-adrenergic receptor/ADRB2 dynamic mass redistribution functional responses to norepinephrine were significantly decreased following CHX treatment, with a time course equivalent to that observed with the SNAP-ADRB2 degradation assay. We subsequently adapted this assay into a 96-well glass-bottom plate format to facilitate high-throughput GPCR degradation screening. t1/2 values quantified for the α1-adrenergic receptor subtypes (ADRA1A, ADRA1B, ADR1D) using the 96-well-plate format correlated with t1/2 values quantified using NIR-PAGE imaging analysis. In summary, this novel assay permits precise quantitative analysis of GPCR degradation in human cells and can be readily adapted to quantify degradation for any membrane protein of interest.

Project description:PURPOSE:Near-infrared fluorescence (NIRF) imaging has been widely used in preclinical studies; however, its low tissue penetration represents a daunting problem for translational clinical imaging of neurodegenerative diseases. The retina is known as an extension of the central nerve system (CNS), and it is widely considered as a window to the brain. Therefore, the retina can be considered as an alternative organ for investigating neurodegenerative diseases, and an eye represents an ideal NIRF imaging organ, due to its minimal opacity. PROCEDURES:NIRF ocular imaging (NIRFOI), for the first time, was explored for imaging of Alzheimer's disease (AD) via utilizing "smart" fluorescent probes CRANAD-X (X = - 2, - 3, - 30, - 58, and - 102) for amyloid beta (Aβ), and CRANAD-61 for reactive oxygen species (ROS). Mice were intravenously injected the fluorescence dyes and images from the eyes were captured with an IVIS imaging system at different time points. RESULTS:All of the tested NIRF probes could be used to differentiate transgenic AD mice and WT mice, and NIRFOI could provide much higher sensitivity for imaging Aβs than NIRF brain imaging did. Our data suggested that NIRFOI could capture the imaging signals from both soluble and insoluble Aβ species. Moreover, we demonstrated that NIRFOI with CRANAD-102 could be used to monitor the therapeutic effects of BACE-1 inhibitor LY2811376. Compared to NIRF brain imaging, NIRFOI provided a larger change of Aβ levels before and after LY2811376 treatment. In addition, we demonstrated that CRANAD-61 could be used to image reactive oxygen species in the eyes. CONCLUSION:The large detection margin by NIRFOI is very important for both diagnosis and therapy response monitoring. Compared to fluorescence microscopic imaging, NIRFOI captures signals with a wide angle (large field of view (FOV)) and can be used to detect soluble Aβs. We believe that NIRFOI has remarkable translational potential for future human studies and can be a potential imaging technology for fast, cheap, accessible, and reliable screening of AD in the future.

Dataset Information

Near-infrared fluorescence imaging of mammalian cells and xenograft tumors with SNAP-tag.

Publications

Near-infrared fluorescence imaging of mammalian cells and xenograft tumors with SNAP-tag.

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