Genetically Engineered “Multicolour” MRI Reporters for In Vivo Imaging of Transgenes Expression
2Department of Biomolecular Sciences, Weizmann institute of Science, Israel
3Department of Chemical Research Support, Weizmann institute of Science, Israel
4Department of Life Sciences Core Facilities, Weizmann institute of Science, Israel
5Russell H. Morgan Department of Radiology, Johns Hopkins University School of Medicine
The ability to map gene expression profiles using fluorescent reporter genes remain a main challenge when it comes to deep tissues. Although the CLARITY approach offers an elegant strategy to clarify tissue opaqueness, more advanced strategies for mapping the expression of multiple (reporter) genes in live organisms is of high demand. We here show the design and implementation of a novel MRI-based approach that allow to map the expression of multiple reporter genes in the brain of live mice.1 Termed GeneREFORM (GENetically Engineered REporters FOR Multicolor-MRI), our approach benefits from the non-invasiveness and unlimited tissue penetration capabilities of MRI, but also allows a color-display of expressed transgenes.
To establish GeneREFORM, the Chemical Exchange Saturation Transfer (CEST) strategy was used to generate pseudo MRI-colors and the PROSS approach was used as an in-silico method for protein-design. Specifically, PROSS was applied to design fully orthogonal genetically engineered multi-substrate enzymes, HSV1-TK and Dm-dNK which specifically convert two MRI-detectable synthetic deoxyribonucleosides (5-MDHT and pdC). As a result, 5-MDHT and pdC that generate MRI distinguishable colors when using CEST, exclusively accumulate in cells expressing the designed reporter genes HSV1-TK_7B and Dm-dNK_7C allowing their spatial display as pseudo-colored MRI maps as demonstrated in tumor model and viral-delivery system (Allouche-Arnon et al. Nat Biotechnol 2022, 40 (7), 1143).
To conclude, given the MRI setup, GeneREFORM has the potential to be applied in a wider range of animal models, including in large animals, and also in clinical setups to accelerate the translation of proposed therapeutics (e.g., gene- and cell-based therapies).