A drug‐selectable acoustic reporter gene system for human cell ultrasound imaging

Abstract A promising new field of genetically encoded ultrasound contrast agents in the form of gas vesicles has recently emerged, which could extend the specificity of medical ultrasound imaging. However, given the delicate genetic nature of how these genes are integrated and expressed, current methods of producing gas vesicle‐expressing mammalian cell lines requires significant cell processing time to establish a clonal/polyclonal line that robustly expresses the gas vesicles sufficiently enough for ultrasound contrast. Here, we describe an inducible and drug‐selectable acoustic reporter gene system that can enable gas vesicle expression in mammalian cell lines, which we demonstrate using HEK293T cells. Our drug‐selectable construct design increases the stability and proportion of cells that successfully integrate all plasmids into their genome, thus reducing the amount of cell processing time required. Additionally, we demonstrate that our drug‐selectable strategy forgoes the need for single‐cell cloning and fluorescence‐activated cell sorting, and that a drug‐selected mixed population is sufficient to generate robust ultrasound contrast. Successful gas vesicle expression was optically and ultrasonically verified, with cells expressing gas vesicles exhibiting an 80% greater signal‐to‐noise ratio compared to negative controls and a 500% greater signal‐to‐noise ratio compared to wild‐type HEK293T cells. This technology presents a new reporter gene paradigm by which ultrasound can be harnessed to visualize specific cell types for applications including cellular reporting and cell therapies.