In a conservation first, a cloned ferret could help save her species | Science

Last month, at a conservation center near Fort Collins, Colorado, staffers held an unusual birthday party, complete with a two-tiered cake made of prairie dog and mouse carcasses, minced meat, and kibble. The recipient of the macabre cake was a small, weasellike animal named Elizabeth Ann. She is the world’s first cloned black-footed ferret, one of North America’s most endangered species, and her first birthday was a major milestone: She is one of the first clones of an endangered species to reach sexual maturity.

Now, Elizabeth Ann—cloned from the cells of a female ferret that died 35 years ago—is poised to make history again. This spring, if all goes as planned, Elizabeth Ann will mate with a carefully selected bachelor in an effort to introduce greater genetic diversity into wild ferret colonies, which are threatened by inbreeding. If she gives birth to healthy kits, it will mark the first time conservation biologists have been able to integrate cloning into an effort to save a species from extinction.

Success could boost nascent efforts to clone other endangered mammals, including rhinos, and help establish the technology as a useful restoration tool. Failure could reinforce long-standing skepticism about the usefulness of cloning in conservation; some researchers see it as too expensive, ethically fraught, and of limited use. They also fear it could distract funders from efforts to address broader issues such as habitat destruction.

“Everything about Elizabeth Ann is much bigger than the science behind it, and it’s much bigger than helping the ferrets,” says Ben Novak, black-footed ferret project lead for Revive & Restore, a nonprofit founded in 2012 to explore how biotechnology might aid endangered and extinct species. “It’s about whether biotechnology can become a part of mainstream conservation.”

The black-footed ferret (Mustela nigripes) is a slender, half-meter-long predator with an ornery streak. It once inhabited huge swaths of the Great Plains, occupying grassland burrows dug by its favorite prey: prairie dogs. By the 1970s, however, the widespread destruction of prairie dog colonies by ranchers, farmers, and others had caused ferret populations to crash. In 1973, the ferret became one of the first species to be designated for protection under the new U.S. Endangered Species Act.

By the late 1970s, the last known ferret colony had disappeared and some biologists believed the species was extinct. But in late 1981, the Wyoming Game and Fish Department received an unexpected call.

One morning a rancher named John Hogg had gone out to investigate the origins of some strange noises heard during the night. He suspected the family dog, Shep, had picked a fight with some varmint. Dead on the ground was a strange, tube-shaped animal that a local taxidermist identified as a black-footed ferret. After wildlife biologists descended on the area around the Hogg ranch, they were overjoyed to discover a good-size colony of more than 100 ferrets.

But within a few years, that colony, too, was in trouble, reduced to just a few dozen animals. In 1985, officials made the difficult decision to round up any ferrets they could find, in hopes of starting a captive breeding program. They captured 18 individuals, but just seven survived to breed, putting the species at risk of inbreeding, which can erode reproductive fitness.

Elizabeth Ann owes her existence to a chance meeting at a banquet in Montana. In 1987, biologist Oliver Ryder was on the lookout for animal cells to freeze. As a young scientist in the 1970s, he had joined a new effort, known as the San Diego Frozen Zoo, that aimed to preserve genetic material from a wide array of endangered mammals, deep-frozen in liquid nitrogen. At a conservation conference, Ryder struck up a conversation with a Wyoming Game and Fish veterinarian named Tom Thorne, who told him about the black-footed ferrets’ plight. A couple of years earlier, Thorne had sent a handful of black-footed ferret tissue samples to the Frozen Zoo, but scientists had managed to preserve a cell line from just one: a male labeled Studbook #2. Ryder suggested Thorne send more. Seven months later, he received skin cells from a single female named Willa, which the zoo also successfully banked.

As those two cell lines sat in a freezer, the ferret breeding program—which has been led by the U.S. Fish and Wildlife Service (FWS) since 1996—began to take off. To date, it has produced some 10,000 ferrets, many of which have been released into the wild. The program has become a prominent success story, but the ferret’s survival is still far from a sure thing.

Black-footed ferret Elizabeth Ann pokes her head out of a tube suspended from the top of a cage.
Elizabeth Ann, the cloned ferret, plays on her first birthday. Soon she will be bred to produce offspring that may invigorate the wild population. USFWS National Black-footed Ferret Conservation Center

One threat is sylvatic plague, a deadly bacterial disease introduced from Asia. Inbreeding has also begun to take a toll on the population, all descendants of just seven animals. Genetic studies, Novak says, show “they’re all superrelated. … The mean kinship falls between that of a sibling and a first cousin.” As a result, they have accumulated some potentially damaging mutations, says genomic researcher Klaus-Peter Koepfli with the Smithsonian Mason School of Conservation. Some animals are born with kinked tails and deformed sternums.

To shore up the ferrets’ genetic diversity, researchers have turned ferret breeding into an exacting science. A computer program helps them assign a desirability rating to each possible pairing, based on the animals’ ancestry. Breeders have also relied on artificial insemination with semen taken in the 1990s from two standout males, known as Scarface and Rocky. But semen is a finite resource, says Pete Gober of FWS, who coordinates the recovery programs.

So, in 2013, program managers turned to Revive & Restore to see whether they could bolster the population’s diversity by transforming the Frozen Zoo’s small vials of preserved cells into living, breathing ferrets. “We wanted to increase and maintain as much genetic diversity as we could from what little amount we had to begin with,” Gober says.

The idea was to use a technology that didn’t exist when the zoo had stored the samples: somatic cell nuclear transfer. In this cloning technology, technicians replace the nucleus of an egg cell with a nucleus taken from a body cell. A jolt of electricity encourages the egg and nucleus to fuse and the cell to multiply; the embryo is then transferred to a surrogate mother. In 1995, Dolly the sheep was the first animal to be born from cloning. Since then, cloning has become a routine tool for duplicating farm animals, pets including cats and dogs, and even prized racing camels.

Cloning for conservation has a far patchier history. When black-footed ferret breeders first approached Revive & Restore, for instance, researchers had created clones of just three endangered species, all hoofed animals: the gaur (Bos gaurus) and European mouflon (Ovis aries musimon) in 2001, and the banteng (B. javanicus) in 2003. In 2015, scientists cloned a fourth endangered species, a sheep known as the Esfahan mouflon (O. gmelini isphahanica). All died fairly young (the banteng lived longest, dying of injuries at age 7), and produced no offspring.

In part, cloning endangered animals has proved harder than duplicating livestock or pets because breeding and husbandry practices are less developed in these species. Conservation programs also have fewer resources than commercial enterprises, so they’re less likely to try again after an unsuccessful attempt.

Cloning endangered species faces unique ethical questions, as well. One is whether the clone, which can hold trace DNA from its surrogate mother, is actually the same as the species that researchers are trying to save. For example, black-footed ferret clones are created using eggs from domestic ferrets, meaning they carry that species’ mitochondrial DNA, which is left in the egg after its nucleus is extracted.

Some conservationists have other concerns. They worry the ability to clone a rare species might undermine support for efforts to protect habitat and keep species alive in the wild. And cloning can be expensive, potentially diverting funds from other conservation activities.

“I think cloning certainly has a future for endangered species, but there are some problems,” says Barbara Durrant, director of reproductive sciences with the San Diego Zoo Wildlife Alliance. For each species, “We will need to be very careful to do the basic research,” she adds.

A few years ago, after extensive technical and ethical reviews, federal regulators decided the potential benefits of cloning the ferrets outweighed the risks. Then, with permits in hand, Revive & Restore teamed up with firms including a pet cloning company called ViaGen Pets and a commercial ferret breeder to develop a plan that cost about $40,000 to execute. It called for creating embryos with DNA taken from Willa, the female black-footed ferret that had died in 1988.

Deepening the gene pool

By cloning a black-footed ferret that died in 1988, biologists hope to add greater genetic diversity to the existing population of this endangered species, which relies heavily on captive breeding for survival.

Breeding a clone of a ferret whose DNA differs from the other remaining ferrets could lead to more genetic variation.
V. Altounian/Science

Even ViaGen’s lead scientist, Shawn Walker, wasn’t sure whether it would work. “We knew we had all the boxes checked,” he says. “But until you hear that heartbeat, you’re always a little bit skeptical, because everything was so new.”

In late 2020, the team implanted Willa-based embryos into three domestic ferrets and shipped them to the National Blackfooted Ferret Conservation Center in Colorado, where about two-thirds of the nation’s captive population lives. To their relief, one ferret gave birth to Elizabeth Ann on 10 December 2020. “It was very exciting,” says Robyn Bortner, captive breeding manager at the facility, who was in the room when the clone was born.

But success still wasn’t a sure thing. The other two pregnancies failed, and Elizabeth Ann had a stillborn litter mate. And when keepers placed Elizabeth Ann with a second surrogate mother and domestic siblings, she often ended up in a dangerous spot: at the bottom of the pile of kits. “It was touch and go the first couple of days. … We kept an incredibly close eye on her,” Bortner recalls. But once Elizabeth Ann’s eyes opened about 1 month later, her black-footed ferret feistiness kicked in. Ever since, Bortner says, “She’s been healthy and everything you would hope.”

Now fully grown, Elizabeth Ann looks and sounds like any other black-footed ferret. She scarfs down meat and stalks and kills live hamsters. (The keepers aren’t willing to risk giving her a live prairie dog, which weighs more than she does.) She chatters angrily at caretakers who get too close. She loves to attack and shred paper bags. And apart from her mitochondrial DNA, most of which comes from her domestic mother, genetic analysis shows she is 100% a blackfooted ferret.

This spring her creators hope to mate Elizabeth Ann with a captive male. Any offspring will still have Elizabeth Ann’s mitochondrial DNA, with traces of domestic ferret. To remove those traces, any male offspring will be paired with captive females, producing kits that no longer carry the domestic female’s mitochondrial DNA.

A tray full of vials with variously-colored caps.
At the San Diego Frozen Zoo, the cells of numerous endangered species are preserved in liquid nitrogen. Cloning a mammal was not yet feasible when the zoo banked cells from two black-footed ferrets in the 1980s.San Diego Zoo Global

Successfully adding Willa’s genes to the black-footed ferret gene pool via Elizabeth Ann would likely “pack this huge biodiversity punch,” Novak says. Genomic analysis has found Willa’s DNA has 10 times more unique alleles than DNA from any captive-bred ferret. That means her chromosomes will “introduce a whole new combination [of traits] and higher level of genetic variants,” Koepfli says. That should slow the ferrets’ trajectory of reproductive decline.

To introduce even more genetic variation into captive-bred ferrets, ViaGen aims to create company for Elizabeth Ann. Scientists there are fine-tuning the cloning procedure to make it more efficient and will try to create the next batch of cloned ferrets in spring 2023, to align with next year’s breeding season.

The successful use of cloning in ferret conservation is likely to attract attention—and perhaps funding—for similar efforts in other endangered species. But replicating it won’t be easy. In part, that’s because species that might benefit from cloning have to meet numerous criteria. The best candidates, for example, have both banked genetic material at the ready and a less endangered close relative that can act as a surrogate. It also helps to have funding and captive breeding infrastructure in place. Few programs can meet these prerequisites—yet.

Another obstacle is that, although the basic cloning process is the same for all mammals, the technology has worked better in some species than others, and “no one knows why,” Durrant says. To increase the odds of success, researchers often must develop a unique “recipe” that addresses an animal’s reproductive quirks, says Samantha Wisely, a conservation geneticist at the University of Florida who works with the black-footed ferret program. “Reproductive technology is super–species specific.”

Still, efforts to clone at least two other endangered species are underway. One is the Przewalski’s horse (Equus ferus przewalskii), a stocky wild horse that once roamed across Europe and Asia. The species nearly went extinct in the mid–20th century, and all individuals alive today are descended from just 12 animals. Luckily, nearly 300 cell lines have been stashed at the Frozen Zoo, and conservationists are now trying to inject some of that lost genetic diversity into the modern population.

In 2020, researchers created Kurt, the clone of a Przewalski’s horse whose cells were frozen 40 years ago. Although he was born several months before Elizabeth Ann, he still has some growing up to do before he’ll be ready to breed. Kurt could be joined by cloned siblings by spring of 2023.

The San Diego Zoo Wildlife Alliance is also in the preliminary stages of trying to clone the northern white rhino (Ceratotherium simum cottoni), the most endangered of several rhino subspecies. Just two living northern white rhinos remain, and neither is capable of giving birth. As a first step, scientists are working on techniques to incorporate the northern white rhino genome into egg cells of a close relative, the southern white rhino (C. simum simum). And once the team hones its techniques, “We certainly hope we can apply them to the black rhino, the Sumatran rhino, and maybe the Javan rhino,” Durrant says.

Some researchers are looking beyond cloning, to other genetic technologies that might help endangered species. Birds, for example, can’t be cloned, but Revive & Restore recently formed a research consortium to develop a technique that could fill a similar role. It involves introducing primordial germ cells from an endangered species into an embryo of a surrogate species, such as a chicken. These germ cells then migrate to the chicken’s gonads and become sex cells. So, a male domestic chicken could produce the sperm of, say, an endangered prairie chicken.

CRISPR gene-editing tools could also play a role in conservation. Editing the genome of black-footed ferrets so that they could resist sylvatic plague, for example, could be a game changer. Koepfli and collaborators are now comparing the black-footed ferret genome with that of its domestic cousin, which plague does not affect, in hopes of identifying the genetic basis of resistance. But actually genetically modifying ferrets and then setting them loose in the wild would require extensive legal and ethical deliberations.

For now, Elizabeth Ann’s caregivers are simply gearing up to pick her ideal first mate. Every male ferret in the six breeding facilities scattered across the United States and Canada is getting intense scrutiny. In part, that’s because Elizabeth Ann is likely to have just a few good breeding years, and annual litters average only three to five kits. Breeders have decided that, first and foremost, they need a proven gentleman—they can’t risk an aggressive ferret hurting their only clone. Excellent genes matter, too. (That criterion could put a male related to Scarface or Rocky in the mix, Novak muses.)

This month, they will develop a short list and make their pick. If the best fit happens to live across the country, they will fly him to Colorado. They’ll collect a semen sample, just in case they need it to artificially inseminate Elizabeth Ann. But they are hoping that once they put the two ferrets together, nature will simply take its course.