Continuous Ultrasound Imaging of Second-Generation Mammalian Acoustic Reporter Genes Expressed Using a Novel Ratio-Tunable Mammalian Polycistronic Expression System.

Ultrasound is an established technology for noninvasive deep-tissue imaging that overcomes the low penetration depth of optical methods. However, until recently it was impossible to couple ultrasound contrast to gene expression or cellular function. Gas vesicles (GVs) were recently introduced as the first genetically encoded ultrasound contrast agents expressed from acoustic reporter genes (ARGs) in bacteria and mammalian cells. However, the first-generation mammalian acoustic reporter genes (mARGs), based on 9 essential GV genes from a soil bacterium B. megaterium, suffer from a number of limitations: linking individual GV genes into compact mammalian polycistronic expression cassettes severely attenuates GV expression and ultrasound contrast compared to the cotransfection of individual genes, both transient and stably transduced mARGs require coexpression of three separate polycistronic cassettes for GV production, and the acoustic properties of B. megaterium GVs only allow imaging using a destructive ultrasound modality, limiting their use to endpoint measurements. Here we present a new generation of mARGs that overcome the above-mentioned limitations. We demonstrate that mARGs2.0 provide strong expression from only two polycistronic expression cassettes with optimized cassette ratio, nonlinear acoustic properties that allow for nondestructive continuous imaging using amplitude modulation pulse sequences, and the ability to modulate acoustic properties based on gene composition. Additionally, we are developing a novel ratio-tunable mammalian polycistronic expression system that enables expression of mARGs2.0 from single transcripts. We extensively characterize mARGs2.0 in vitro and demonstrate their utility in vivo by imaging gene expression in an orthotopic cancer model. For this purpose, we stably integrated these reporter genes into the genomes of human breast cancer cells and imaged them nondestructively in vivo after tumor formation in mouse mammary fat pads and chemically induced expression.