Optical clearing for fluorescence visualization of dCas9/FP expression in tumor subcutaneous xenografts in mice
The goal of our study was to detect the dCas9 orthologs expression under an inducible promoter to ensure regulated expression of chimeras in tumor animal models using fluorescence imaging with optical clearing.
A549 cells were established for expressing double chimeras (SttdCas9-EGFP and NmdCas9-mCherry, clone E9) under inducible Tet-O doxycycline promoter in according to Tumor xenografts were established and maintained in athymic mice that were given doxycycline via gavage followed by fluorescence imaging using a planar system. It was shown that the highest fluorescence signal in tumor xenografts was registered on the 3rd day after induction of chimeric protein expression. Subsequently fluorescence detection was carried out on the 3rd day. To improve the contrast, a 0.7 M solution of gadobutrol was used. FI of red chimera expressed in tumor xenograft was amplified two-fold in vivo by applying 0.7 M gadobutrol due to the optical clearing (OC) of the skin. The MRI study reflected the perfusion of the tumor and coincided with the area of fluorescence.
The use of optical clearing approach enabled high-contrast imaging of dual (red and green fluorescent) chimeric dCas9- based proteins expression in tumor xenografts have been demonstrated. MR contrast agent gadobutrol improved both the intensity and contrast of FI as well as mapping of tumor perfusion by MRI.
The work was supported by the grant of Russian Science Foundation № 22-14-00205 https://rscf.ru/project/22-14-00205/
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Research Center of Biotechnology/Vavilov Institute of General Genetics of the RAS
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