Expectant scientists hoping to catch a glimpse of gene expression inside living cells are often disappointed by conventional imaging technology, which relies on luminescent or fluorescent reporter genes. Though they glow brightly when they are expressed along with genes of interest, reporter genes often remain hidden. Their light cannot penetrate most living tissue. Fortunately, imaging technology doesn’t have to rely on optical signals. It can take advantage of acoustic signals, which are more penetrating.

A nascent imaging technology conceived and developed by Caltech scientists has the potential to reveal gene expression deep inside the body. The technology, which borrows genes from buoyant bacteria, allows mammalian cells to produce tiny air-filled protein compartments that may serve as ultrasound contrast agents. Ultrasound, as is well known, can penetrate deeply through tissue.

Although the idea of using ultrasound to image gene expression in live cells is elegant, getting the idea to work proved awkward. It required that nine genes be transplanted from bacteria into mammalian cells—in this case, cells derived from human kidneys.

Resolving all the difficulties took several years, the Caltech team reported. They summarized their work in a paper (“Ultrasound imaging of gene expression in mammalian cells”) that appeared September 27 in Science.

“The translation machinery is very different in the two kinds of cells,” said graduate student Arash Farhadi, the paper’s lead author. “One of the biggest differences is that in bacteria it is common to have multiple genes arranged in the DNA such that they are transcribed into one shared piece of RNA, which is then translated into all the corresponding proteins, whereas in eukaryotes, every gene is usually on its own.”

To get around this difficulty in using bacterial DNA, the Caltech team borrowed from yet another source of DNA: viruses. “Viruses also need to trick mammalian cells into expressing a bunch of proteins,” said Mikhail Shapiro, PhD, a professor of chemical engineering and the senior author of the current study. “So, we used viral elements to trick the cell into producing multiple genes from a shared piece of RNA.” In this way, Farhadi and colleagues combined eight genes together on a single piece of RNA.

However, even after the bacterial DNA was installed in the HEK cells and functioning, the work was incomplete. The cells were making gas vesicle proteins, but gas vesicles were not forming. It turned out that proteins not only needed to be produced, but in the right ratios.

Shapiro likened the assembly of gas vesicles to a construction project. A building might be made of wood, glass, and bricks, but if workers show up with mostly windows and only a few bricks, they will not be able to construct a building.

In addition to providing the building materials, some proteins encoded by the gas vesicle genes act like construction machinery—the cranes, bulldozers, etc.—that are used to make the gas vesicles. If a construction site has 50 cranes but only one bulldozer, the project probably will not get finished. Ratios, again, are key.

“The correct ratios of proteins are programmed into the bacterial gene clusters,” noted Farhadi, “but when we put them into the mammalian cells, we have to figure out what those ratios need to be and how to get the mammalian cells to make those correctly.”

Eventually, the Caltech scientists succeeded in optimizing their genetic constructs, and they presented the results of their proof-of-principle experiments.

“Our results establish the ability of an engineered genetic construct encoding prokaryote-derived gas vesicles to serve as a mammalian reporter gene for ultrasound, providing the ability to monitor cellular location and function inside living organisms,” the authors of the Science article wrote. “Mammalian acoustic reporter genes, or mARGs, provide many of the capabilities associated with established genetically encoded optical reporters, including imaging cellular dynamics by promoter-driven expression and mapping cellular populations in complex samples.”

According to the paper, mARGs allow cells to be visualized at volumetric densities below 0.5% and permit high-resolution imaging of gene expression in living animals.

The authors added that although their genetic constructs should be immediately useful in a variety of contexts, considerable scope exists for further optimization to make mARGs as widely useful as green fluorescent protein. “There has been more than 20 years of work improving fluorescent proteins,” noted Shapiro, “and we probably have 20 years of work to improve what we’ve developed, but this is a key proof of concept.”