The last known Pyrenean ibex, a wild goat named Celia, died more than two decades ago, the victim of a falling branch. But before she died, scientists managed to biopsy her skin and stash the sample in a freezer. They were already envisioning a future in which cloning might enable geneticists to bring species back to life.

In 2003, they thawed those cells and made a first attempt to clone Celia. Since they didn’t have any living Pyrenean ibex, they had to get creative. They removed genetic material from goat eggs and replaced it with DNA from Celia’s skin cells. After a mild electric shock, the eggs began to divide. The scientists then implanted these embryos into surrogate moms — goats or goat hybrids. This process — known as interspecies cloning — is tricky. One kid made it to term, but he died a few minutes after he was born.

Francisco Pelegrí first learned about the ibex cloning effort when one of his students brought a news article describing the feat to class. Pelegrí, a geneticist, was stunned. “From a technical perspective, it didn’t make sense,” he says. “By the time you only have 100 individuals, you’re pretty close to extinction.” The researchers had cells from a single animal, and they were trying to bring an entire species back. “It struck me that we really are not prepared for this at all,” Pelegrí says.

The Earth is in the midst of a sixth mass extinction event, and most scientists point to human activity as the primary cause. Each day, the planet loses an average of five to 30 species. While efforts are under way to preserve their habitat, these efforts may not be enough to save them. Extinct species, by definition, no longer exist. But their genetic material can live on in biobanks, offering the possibility of resurrection. Think of it as an extinction loophole.

Pelegrí thinks this loophole will become an increasingly crucial part of conservation. But to successfully leverage it, scientists need a smarter way to proactively biobank samples — not just one or two, but hundreds, from each species. They also need to understand the rules that govern interspecies cloning. For example, how close do two species need to be on the tree of life for cloning to succeed? With a grant from UW2020, a campus-based initiative that rewards high-risk, high-impact research, Pelegrí and his collaborators aim to find out.

“Everybody agrees we need seed banks for plants, but when we talk about seed banks for animals, people start to think it’s crazy,” Pelegrí says. But to him, it seems like an obvious and necessary solution to curb the catastrophic loss of biodiversity. “Everything revolves around climate change and population control. We’re doing our best to provide all the tools that we’re going to have at our disposal to help with the problem.”

Animal Lover

Pelegrí has always had a passion for nature. As a child growing up in Venezuela, he watched nature shows hosted by the Spanish naturalist Félix Rodríguez de la Fuente and developed a deep love of animals. He memorized their scientific names and collected trading cards with their pictures.

When Pelegrí bought a small farm near Madison in 2006, he began thinking about what kind of animal he should raise to support conservation. He landed on a breed of endangered ponies. “They’re essentially the only derivatives we have of the European wild horses, which became extinct,” he says.

At the time, conservation was a hobby. In the lab, Pelegrí focused on developmental genetics, trying to work out how an egg becomes an embryo. “My passion for nature has always been there, but it was not a part of my profession,” he says.

After Pelegrí read the story about the ibex, however, he started learning more about the role of genetics in conservation. But, as he read more about the field, he grew increasingly bewildered. Scientists seemed to be jamming genetic material from one animal into another willy-nilly — whale into pig egg or panda into rabbit egg. These mash-ups — called cybrids — didn’t have a chance at succeeding. “The organisms were so far apart,” he says.

Pelegrí realized he could use his two decades of expertise in developmental genetics and his own zebrafish lab to work out which pairings could be successful and which could not. “I can literally apply what I’ve learned in all these decades to something that I’ve always cared about,” he says.

Fish Family

In the basement of the UW Biotechnology Center, Pelegrí pushes open a gray steel door to reveal row after row of shelves stacked with glass and plastic tanks. Each tank houses dozens of zebrafish — Danio rerio. The main room and a smaller back room currently hold some 50,000 fish. Pelegrí, in bright green Crocs and a navy “Badger State” hoodie, points out some that glow pink and babies no bigger than the tip of a pencil.

Zebrafish aren’t endangered, of course. They’re readily available in pet stores and labs all around the globe. But some of their distant cousins are under threat. Pelegrí believes that he and his colleagues can use this family of fish to work out the limits of interspecies cloning.

At the front of the fish room, Ryan Trevena, a graduate student in Pelegrí’s lab, is busy matchmaking, pairing males and females and placing them in small tanks. The females’ bellies are swollen with eggs. As soon as one of these fish begins to lay, Trevena scoops her out and places her in a beaker of anesthetic to knock her out. He carefully dries her with a paper towel and then gently presses on her belly with a gloved finger until a drop of milky liquid appears on her abdomen. This single droplet carries dozens of microscopic eggs. He treats the eggs with a chemical that, with help from a UV light, breaks down their nuclei.

Upstairs, in a much smaller room, dozens of tanks hold other members of the Danionin family. Some have Dalmatian-like spots. Others are almost translucent. Trevena mixes the zebrafish eggs he just collected with sperm from Danio albolineatus, the pearl danio. Over the next several hours, the eggs will start dividing. Three days later, they will hatch.

These fish won’t survive — they have only half the genetic material of a normal fish and typically die in the first few hours after birth. But it’s enough time for Pelegrí and his colleagues to examine whether cloning the pearl danio using a zebrafish egg might work. Performing nuclear transplants is finicky work that takes time, “and the success rate is fairly low,” says genetics Ph.D. student Trevor Chamberlain MS’19, who is working on the project with Pelegrí and Trevena. In vitro fertilization — the process they’re using — is a quick and easy way to screen for combinations that might work. “We’re doing it for the through-put,” Chamberlain says.

For cloning to work, a transplanted nucleus must communicate with the rest of the egg cell. If the nucleus and egg are from two different species — especially distantly related ones — that communication can break down. For example, “the mitochondria only code for a small handful of genes, but they’re core genes,” Chamberlain says. They are key to respiration. If the mitochondrial DNA and the nuclear DNA aren’t compatible, the embryo may never develop correctly. In primates, humans can mix with chimpanzees, Pelegrí says. “But when you get as far as orangutans, then it breaks down.”

Knowing these limits will be essential if biologists want to boost endangered populations or revive extinct animals through cloning. Using eggs from the animal’s closest living relative might produce the best success, but it won’t always be feasible.

One model system, of course, won’t be enough. The limits that exist in the Danionin lineage may not apply to other families. “We need to look at other lineages precisely for that reason,” Pelegrí says. That’s why he has recruited collaborators working with other model organisms: frogs and bees.

Brilliantly colored mantella frogs, from Madagascar, are tiny and poisonous. Eleven species are either at risk, endangered, or vulnerable. The golden mantella, which now exists only in one small patch of forest, is critically endangered. And many other amphibians are under threat too from a fungal outbreak that has decimated populations around the globe. According to a 2019 study, fungi contributed to the decline of some 500 species between 1965 and 2015. Of those, 90 are presumed extinct.

Pollinator populations have also been on the decline. “These insects are critically important, so their declines are pretty troubling,” says Sean Schoville, a molecular ecologist, associate professor of entomology, and Pelegrí’s collaborator. “We are focusing on bees — mostly bumblebees — as a good model because they’re declining across the world, but especially in the United States,” he says. “There are more direct measures of conservation that are still possible with insects. But we might find ourselves needing these kinds of techniques because we haven’t actually found the cause of the decline.”

The factors that create a mismatch between nucleus and egg won’t be the same for every family. The boundaries might be different for bees than they are for fish or frogs. But by studying all three groups, Pelegrí says, the team might be able to “get a ballpark idea of what those parameters might be.”

Cloning is one way to revive endangered populations or re-create extinct ones, but there are other ways that might work better. One method being considered for mammals relies on the plasticity of the mammalian embryo. In the earliest stages, scientists can fuse cells from an endangered species onto the embryo. That organism then becomes a chimera — part engendered, part not. And some of its germ cells might be wholly composed of endangered species DNA.

“You could get sperm that is pure sperm from an endangered species or pure eggs from endangered species,” Pelegrí says. Those cells could then be mixed to create an embryo that is wholly the endangered species.

Take It to the Bank

The success of cloning as a conservation strategy depends, in large part, on having well-stocked, long-lasting biobanks. Pelegrí envisions a network of biobanks that would house samples from thousands — or even tens of thousands — of species. To maintain genetic diversity, they would store samples from 500 individuals for each one. The first conundrum is how to obtain samples from species that are already under threat. “You cannot go somewhere and be invasive and affect the species you want to save,” Pelegrí says.

One idea is to outsource the collection to mosquitoes. Their guts hold blood from a wide variety of species, “a possum or a tiger or whatever,” Pelegrí says. If researchers can catch the insects and identify which cells belong to which animal, this method could be an easy, noninvasive way to get cells from species whose populations are already dwindling. (Read about how students are engaging in sample collecting and learning about conservation genetics through a study abroad course in Costa Rica in Research Creates Teaching Opportunities.)

A second hurdle is the biobanking itself. Existing animal biobanks store cells in massive subzero freezers, “which, just from an energy standpoint, is pretty costly,” says Caroline Barry BS’16, a graduate student who is working with Pelegrí. It also makes them vulnerable to power outages or political whims. So the team is working to develop less energy-intensive ways to preserve samples. The goal is to make the animal cells more like plant seeds.

Barry hopes to do that by taking some lessons from the adorable and nearly indestructible tardigrade (also known as the water bear or moss piglet). These animals can survive for years, or even decades, without water. They can withstand blasts of UV radiation, extreme temperatures, and the vacuum of space.

Barry is currently trying to grow tardigrades in the lab, and then she’ll begin working to imbue fish eggs with some of the tardigrade’s toughness by bathing them in tardigrade messenger RNA. That might allow for eggs to be stored at higher temperatures. And if the tardigrade doesn’t work, there are plenty of other bio-inspired options to explore. Keratin — a protein found in hair, nails, feathers, and horns — might provide good protection from bacteria and help keep DNA stable. Or the team might be able to use cells called osteoblasts, which play a crucial role in bone formation, to encase the samples in a tough mineral shell. They are using new mRNA delivery methods, which allow them to test these different options efficiently.

Barry is also investigating methods for giving individual samples unique barcodes so that entire populations can be stored in a single vial. Vertebrates alone account for 66,000 species. So combining individuals would save much-needed space.

Conservation biologists tend to view cloning and other genetic manipulations as a last-ditch effort to save species. But Pelegrí says it’s crucial to be proactive rather than reactive. Samples need to be collected and banked before populations begin to crash. “We don’t have to wait until the species become extinct to do anything,” he says. “In fact, we shouldn’t.” As a population shrinks, so does its genetic diversity. Cloning could be used to reinject diversity, essentially boosting a struggling species.

“These technologies are coming,” Pelegrí says. In 50 years, they might be commonplace. “What we need to do now is prepare for that future.”

 

A Cross-Campus Collaboration

The animal biodiversity biobanking project involves faculty, staff, and students from the College of Agricultural and Life Sciences, the College of Engineering, the College of Letters and Science, the Morgridge Institute for Research, the Nelson Institute for Environmental Studies, and the School of Medicine and Public Health. Here are some of the key collaborators.

Principal Investigator: Francisco Pelegrí, professor of genetics

Co-Principal Investigators: Elizabeth Hennessy, assistant professor of history; William Murphy, associate professor of biomedical engineering; Paul Robbins, director, Nelson Institute for Environmental Studies

Co-Investigators: Wesley Culberson, assistant professor of medical physics; Claudio Gratton, professor of entomology; Susan Paskewitz, professor and chair of entomology; Sean Schoville, assistant professor of entomology; James Thomson, director of regenerative biology, Morgridge Institute for Research