Program

PO4-1-50

Development of a tag-probe based cell labeling technique for in vivo fluorescence imaging

[Speaker] Hiroyuki Okamoto:1
[Co-author] Daisuke Asanuma:1,2, Shigeyuki Namiki:1, Kenzo Hirose:1
1:Neurobiology, Graduate School of Medicine, The University of Tokyo, Japan, 2:PREST, JST, Japan

Background: Near-infrared (NIR) fluorescence (λ: 700-900 nm) is very useful for in vivo imaging, because it has high tissue permeability and less contamination of autofluorescence. NIR fluorescent proteins, such as IFP and iRFP, have been developed, but their low brightness is a drawback in in vivo imaging. On the other hand, chemical biology techniques, such as HaloTag labeling technique, enable labeling of cellular molecules of interest with small-molecular, bright NIR fluorescent dyes. However, non-specific staining by unlabeled dyes often compromises their applications to in vivo imaging. Our goal is to develop a new chemical biology technique for specific cell labeling using NIR fluorescence in living animals.
Methods: To develop our technique, we synthesized small-molecular QODE (Quenched Organic Dye Emission) probe and obtained a single-chain variable fragment (scFv), named as DeQODEtag (DeQuenching Organic Dye Emission tag), by a conventional method of mouse immunization, hybridoma screening and subsequent antibody engineering. We evaluated in vitro fluorescence properties of QODE probe. DeQODEtag was introduced into SKOV3 cells by viral infection. QODE probe was applied to DeQODEtag-expressing SKOV3 cells and fluorescence imaging was performed. We prepared mouse models bearing subcutaneously implanted DeQODEtag-expressing and non-expressing SKOV3 cells, and performed in vivo imaging of the mice with intravenously administered QODE probe.
Results: We designed DeQODEtag technique, in which QODE probe composed of an NIR fluorescent dye and a quencher is almost non-fluorescent but it becomes fluorescent by binding to quencher-binding DeQODEtag. We synthetically developed QODE probe, adopting Si-rhodamine as a dye and dinitrophenyl group (DNP) as a quencher. We also obtained an anti-DNP scFv, DeQODEtag. In in vitro experiment, the fluorescence intensity of QODE probe was increased upon addition of DeQODEtag by 77-fold at the peak emission wavelength of 719 nm. In live-cell imaging experiment, we confirmed specific staining of DeQODEtag-expressing SKOV3 cells by QODE probe. In in vivo imaging experiment, QODE probe specifically labeled DeQODEtag-expressing SKOV3 cells in mouse models bearing subcutaneously implanted DeQODEtag-expressing and non-expressing SKOV3 cells.
Conclusions: DeQODEtag technique successfully performed specific cell labeling using NIR fluorescence in living animals. Future work will focus on applications of this technique to in vivo brain imaging.
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