CC BY
2*
ALT'23 The 30th International Conference on Advanced Laser Technologies
B-I-22
Registration of low-intensity fluorescence in subcutaneous xenografts: from problems to their solutions
Victoria Zherdeva1, Alexei Bogdanov Jr 4, Gerel Abushinova 3' Lylia Maloshenok 1 2,
1-Research Center of Biotechnology RAN, AN Bach Institute of Biochemistry, Moscow, Russian Federation 2- Vavilov Institute of General Genetics RAN, Moscow, Russian Federation 3- Moscow State University, Biology, Moscow, Russian Federation 4- UMASS Chan Medical School, Worcester, United States of America
Various strategies for imaging living cells altered by genome editing with CRISPR/Cas9 system have been implemented since discovery [1]. Catalytically inactive Cas9 (dCas9) has multiple applications with the most useful being the activation/repression of transcription [2]. The use of dCas9 may assist in mapping of genes within chromatin structure at the level of individual cells and intact tissue. The goal of our study was in developing and testing of dCas9 orthologs expressed under an inducible promoter to ensure regulated expression of chimeras in cells and in tumor animal models.
All chimeric constructs were cloned into 3d-generation lentiviral (LV) transfer vector FU-tet-o-hOct4 with Tet-On doxycycline (Dox) -regulated gene expression. dCas9 orthologs from S. thermophilus (Stt) and N. meningitides (Nm) and fluorescent proteins (FP) were fused with 2 NLS at 1:1 ratio. A pair EGFP and mCherry were selected as FPs due to their potential to engage in emissive FRET. The obtained transfer vectors (FU-Tet-o-SttdCas9-EGFP and FU-Tet-o-NmdCas9-mCherry) were used for LV particles production and HEK293T and A549 cells transduction for establish clones expressing double chimeras (SttdCas9-EGFP and NmdCas9-mCherry, E9). Tumor xenografts were maintained in athymic mice that were given doxycycline via gavage followed by fluorescence imaging using a planar system.
Cells expressing Stt1 dCas9-EGFP and Nm dCas9-mCherry showed normal morphology with predominantly nuclear dual fluorescence. Before FP chimera induction with Dox there was no fluorescent signal noted in cells showing tight regulation of chimera expression. FP expression in vivo was observed 1 day post induction and fluorescent signal underwent a decrease during the course of 4-5 days if Dox was withdrawn. 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 according to [4]. 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. Conclusion
The use of optical clearing approach enabled high-contrast imaging of dual (red and green fluorescent) chi-meric 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.
Acknowledgement
The work of V. Zh., L.M., G.A. was supported by the grant of Russian Science Foundation № 22-14-00205 https://rscf.ru/project/22-14-00205/ The authors declare no competing financial interest. References
[1] Ma H. et al. 2015. Multicolor CRISPR labeling of chromosomal loci in human cells. Proc. Natl. Acad. Sci. U. S. A. 112: 3002-3007.
[2] Gao Y. et al. 2016. Complex transcriptional modulation with orthogonal and inducible dCas9 regulators. Nat. Methods. 13:1043-1049.
[3] Cawthorne C. et al. 2007. Comparison of doxycycline delivery methods for Tet-Inducible gene expression in a subcutaneous xenograft model. J. Biomol. Tech. 18:120-123.
[4] Kazachkina NI, et al. 2021. Topical cutaneous gadobutrol application causes fluorescence intensity change in RFP-expressing tumor bearing mice. J. Biomed Phot & Eng. 7: 02030.
[5] Maloshenok, L et al. 2023. Tet-Regulated Expression and Optical Clearing for In Vivo Visualization of Genetically Encoded Chimeric dCas9/Fluorescent Protein Probes. Materials. 3, 16, 940.
