UC Santa Barbara

Molecular phenomena such as gene expression, protein-protein interactions, and the influence of pH, ion movement, and endogenous small molecules for cellular function have grown with increasing interest since the discovery of fluorescent proteins (FPs) in the 1960s and further development as a genetic reporter shortly after. The ability to precisely image “invisible” proteins by tagging them with FPs has revolutionized the field of molecular biology. Although FPs have immensely increased our understanding of biomolecular events, visualizing these same biological processes beyond cell cultures and into higher order mammals is greatly hindered due to photon absorption and scattering from tissue, severely limiting light penetration to a mere millimeter. Due to these limitations, engineering a genetic reporter to monitor gene expression in deeper-seated tissue would greatly benefit the scientific community to study biomolecular phenomena in this context. Magnetic resonance imaging (MRI) based genetic reporters can help complement their fluorescent counterparts in deep tissue imaging with relatively high spatial resolution (~250 µm) and temporal resolution (<1s). However, current MRI reporters developed in the past 20 years are prone to lower sensitivity in the presence of physiological pathologies and abnormalities, thereby convoluting signal contrast. Herein, we establish human aquaporin-1 (AQP1) as a viable MRI reporter that can functionally produce a switch-like signal upon chemical administration at the post-translational level. Post processing of the images obtained before and after ligand addition subtracted out endogenous background noise contributions enhancing reporter signal. We measured the response of 7 ligand-responsive degradation tags fused to either the N or C terminus of AQP1 conferring AQP1-dependent contrast in chinese hamster ovary tetracycline on (CHO tet ON) cells. Thereafter, we translated the best 2-3 tags to measure their performance in human glioblastoma U87 cells, human acute T-cell leukemia Jurkat cells, and murine macrophage RAW 264.7 cells demonstrating this reporter system in diverse cell types. Furthermore, we applied this reporter system in immunocompromised NOD scid gamma (NSG) mice using xenografted flank tumors demonstrating on-demand MRI signal in deeper-seated tissue. These results provide a novel proof of concept for obtaining higher MRI sensitivity in combination with gene reporting in physiological environments that have previously been difficult to detect.