Search

Tracking gene expression in deep tissues requires genetic reporters that can be unambiguously detected using tissue penetrant techniques. Magnetic resonance imaging (MRI) is uniquely suited for this purpose; however, there is a dearth of reporters that can be reliably linked to gene expression with minimal interference from background tissue signals. Here, we present a conceptually new method for generating background-subtracted, drug-gated, multiplex images of gene expression using MRI. Specifically, we engineered chemically erasable reporters consisting of a water channel, aquaporin-1, fused to destabilizing domains, which are stabilized by binding to cell-permeable small-molecule ligands. We showed that this approach allows for highly specific detection of gene expression through differential imaging. In addition, by engineering destabilized aquaporin-1 variants with orthogonal ligand requirements, it is possible to distinguish distinct subpopulations of cells in mixed cultures. Finally, we demonstrated this approach in a mouse tumor model through differential imaging of gene expression with minimal background.

Aquaporin-1 (Aqp1), a water channel, has garnered significant interest for cell-based medicine and in vivo synthetic biology due to its ability to be genetically encoded to produce magnetic resonance signals by increasing the rate of water diffusion in cells. However, concerns regarding the effects of Aqp1 overexpression and increased membrane diffusivity on cell physiology have limited its widespread use as a deep-tissue reporter. In this study, we present evidence that Aqp1 generates strong diffusion-based magnetic resonance signals without adversely affecting cell viability or morphology in diverse cell lines derived from mice and humans. Our findings indicate that Aqp1 overexpression does not induce ER stress, which is frequently associated with heterologous expression of membrane proteins. Furthermore, we observed that Aqp1 expression had no detrimental effects on native biological activities, such as phagocytosis, immune response, insulin secretion, and tumor cell migration in the analyzed cell lines. These findings should serve to alleviate any lingering safety concerns regarding the utilization of Aqp1 as a genetic reporter and should foster its broader application as a noninvasive reporter for in vivo studies.

Calcium-responsive contrast agents for magnetic resonance imaging (MRI) offer a promising approach for noninvasive brain-wide monitoring of neural activity at any arbitrary depth. Current examples of MRI-based calcium probes involve synthetic molecules and nanoparticles, which cannot be used to examine calcium signaling in a genetically encoded form. Here, we describe a new MRI sensor for calcium, based entirely on a naturally occurring calcium-binding protein known as calprotectin. Calcium-binding causes calprotectin to sequester manganese ions, thereby limiting Mn2+ enhanced paramagnetic relaxation of nearby water molecules. We demonstrate that this mechanism allows calprotectin to alter T1 and T2 based MRI signals in response to biologically relevant calcium concentrations. The resulting response amplitude, i.e., change in relaxation time, is comparable to existing MRI-based calcium sensors as well as other reported protein-based MRI sensors. As a preliminary demonstration of its biological applicability, we used calprotectin to detect calcium in a lysed hippocampal cell preparation as well as in intact Chinese hamster ovary cells treated with a calcium ionophore. Calprotectin thus represents a promising path toward noninvasive imaging of calcium signaling by combining the molecular and cellular specificity of genetically encodable tools with the ability of MRI to image through scattering tissue of any size and depth.

Calcium ions represent one of the key second messengers accompanying neural activity and synaptic signaling. Accordingly, dynamic imaging of calcium fluctuations in living organisms represents a cornerstone technology for discovering neural mechanisms that underlie memory, determine behavior, and modulate emotional states as well as how these mechanisms are perturbed by neurological disease and brain injury. While optical technologies are well established for high resolution imaging of calcium dynamics, physical limits on light penetration hinder their application for whole-brain imaging in intact vertebrates. Unlike optics, magnetic resonance imaging (MRI) enables noninvasive large-scale imaging across vertebrates of all sizes. This has motivated the development of several sensors that leverage innovative physicochemical mechanisms to sensitize MRI contrast to intracellular and extracellular changes in calcium. Here, we review the current state-of-the-art in MRI-based calcium sensors, focusing on fundamental aspects of sensor performance, in vivo applications, and challenges related to sensitivity. We also highlight how innovations at the intersection of reporter gene technology and gene delivery open potential opportunities for mapping calcium activity in genetically targeted cells, complementing the benefits of small molecule probes and nanoparticle sensors.