When the pile of opossums arrived at John VandeBerg’s lab from the Smithsonian’s National Zoo in 1978, the geneticist had an ambitious plan for the soft-eyed, hamster-sized animals. He wanted to domesticate them to live in a lab anywhere on the planet. Mice were well and good, but imagine what biomedical insights might be lurking inside marsupials, he thought. Their young, rather than being encased inside a uterus, develop attached to a nipple in a pouch or on a belly where they’re much easier to observe.
VandeBerg succeeded, and today manages the largest Monodelphis domestica colony in the world at the University of Texas Rio Grande Valley School of Medicine. More than a dozen labs have taken up research projects with the gray short-tailed opossums, who hail from South America. But the same quirk that makes them a compelling model organism has also made them more difficult to study than he once imagined.
Shortly after ovulation, marsupial moms secrete layers of mucus and protein around their eggs, covering them in a hard, protective shell. As a result, unlike the mouse, whose jelly-ensconced genome readily yielded to manipulation, opossum DNA was effectively locked away, far from scientists’ probing pipettes. Throughout the ’90s and 2000s, lab after lab tried and failed to make designer opossums that would make it possible to study the function of different genes. They just couldn’t get the needle through the shell. “I never thought I would see the day when a transgenic laboratory opossum would be produced,” VandeBerg told STAT. But today, to his delight, he was proved wrong.
A group of researchers from the RIKEN Center for Biosystems Dynamics Research in Japan reported Wednesday that through a series of tweaks to traditional opossum rearing combined with a new method for delivering the gene-editing tool CRISPR to embryos, they had overcome this decades-old impediment. Using their techniques, they created litters of albino pups lacking a gene necessary for developing fur and eye pigmentation. The study, published in Current Biology, marks the first time anyone has ever genetically engineered a marsupial.
The advance promises to unlock new insights into human biology and disease, aiding in the study of everything from the developing immune system to tissue regeneration to skin cancer.
“Studying biodiversity is not just about exploring the biology of a bunch of interesting organisms, but ultimately for a better understanding of human biology,” developmental biologist and lead study author Hiroshi Kiyonari said via email.
Five years ago, his team began to systematically work out the problem that had so long plagued the opossum field. The first barrier was to collect zygotes (fertilized eggs) at the right time. Ideally, that would be before they began dividing, when they are still a single cell. If you inject CRISPR at this stage, you can be sure all the resulting animals’ cells will carry whatever DNA changes you make. Doing it later can mean some cells but not others will be edited — a less ideal outcome known as mosaicism. Another benefit of collecting fertilized eggs as early as possible is that the shell coat hasn’t had time to thicken.
But opossums are unpredictable. Put a male and female together, and they might take a week or two to get down to business. So Kiyonari’s teams set up infrared cameras in the lab to track their every amorous movement. They also administered hormones to females to induce estrus and optimized the lab’s light-dark schedule to make breeding more predictable. That yielded a collection of embryos between the one- and four-cell stage.
The next technical hurdle was actually injecting CRISPR. Researchers on Kiyonari’s team had previously developed a technique in mice and rats that they thought might work with opossums. It involved using an apparatus called a piezoelectric actuator, which converts a jolt of electricity into a physical force. The actuator advances the injection needle repeatedly by a very short distance at very high speeds. “That made it possible for us to perform microinjection into opossum zygotes without significant damage, and was one of the keys to success,” wrote Kiyonari.
Because this opossum species develops melanomas much the same way humans do, and because their young can regenerate a severed spinal cord, scientists hope that this new ability to manipulate its genome will boost research into treating human skin cancer and neurological ailments.
Better tools for studying a distant evolutionary cousin might reveal other insights, too.
In 2006, Rob Miller, a biologist at the University of New Mexico, helped sequence the first marsupial genome: Monodelphis domestica. Later, when examining the regions that coded for the animals’ immune cells, he and his team discovered a strange-looking gene — it appeared to be one part T cell receptor, one part antibody. All mammals have just two kinds of T cells, based on the type of receptor they have. This would have been a third. They kept investigating, but it was slow going.
“The difficulty of working with an animal like this, even though it’s easy to keep in captivity, is there aren’t a lot of tools,” said Miller. Eventually, through painstaking collection of single-cell data, they confirmed their hunch. Their discovery of this ancient T cell lineage that other mammals lost sometime after they split off from marsupials was published in Science in March. Now comes the harder work of figuring out what these T cells do. And that’s why Miller is so enthusiastic about the CRISPR’d opossums.
“The obvious thing to do would be to knock out these T cell receptor genes and watch opossums that don’t have them grow up and see what happens,” said Miller. But until now there hasn’t been a way to do that. “We’ve been finding lots of interesting genes that marsupials have that humans don’t — many of them in the immune system — whose function remains unknown because we don’t have a way of manipulating them. These guys just produced the potential ability to do that, so it’s hugely exciting.”
VandeBerg also believes the advance could be transformative. For years, he’s been in conversations with the National Institutes of Health about establishing a national center for opossum research, similar to the network of National Primate Research Centers established by Congress in the early 1960s for advancing biomedical research. Such a center could house a large colony and support researchers around the country. With this latest advance, it could also serve as a central resource for creating different genetically modified lines of animals. The idea has yet to gain traction, but VandeBerg thinks with more tools available, the value of such a center would only increase.
“I expect we’ll see many papers using this technology coming from Japanese groups over the next few years,” he said. “And if the United States does not develop its own capacity, we’re really going to be left behind in this exciting area of biomedical research.”