Last month, at a conservation center near Fort Collins, Colorado, employees hosted an unusual birthday party with a two-tiered prairie dog and mouse carcass cake. The recipient of the creepy cake was a small, weasel-like animal named Elizabeth Ann. She is the world's first cloned black-footed ferret, one of North America's most rapidly endangered species, and her first birthday was a 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 ready to make history again. This spring, if all goes according to plan, Elizabeth Ann will mate with a carefully selected bachelor to bring more genetic diversity to a colony of wild ferrets threatened by inbreeding. If she gives birth to healthy puppies, it will be the first time biologists have been able to integrate cloning technology into efforts to save a species from extinction.
The success could spur nascent efforts to clone other endangered mammals, including rhinos, and help make the technology a useful tool in efforts to conserve the species. But failure in this area may reinforce long-standing skepticism about the usefulness of cloning, and some researchers see it as too expensive, unethical and limited to use. They also fear that it could detract from efforts to address broader issues such as the destruction of habitat ecosystems.
“Everything about Elizabeth Ann is so much more than the science behind it, and it’s so much more than helping ferrets,” says Ben Novak, Project Manager for the Black-footed Ferret at Revive & Restore, a non-profit organization founded in 2012 to explore how biotechnology can help endangered and extinct species. It is about whether the progress made in this area can become part of the conservation mainstream.
The black-footed ferret (Mustel nigripes) is a graceful half-meter predator with a vicious disposition. It once roamed the vast expanses of the Great Plains, burrowing in pastures dug by its favorite prey: prairie dogs. However, by the 1970s, mass destruction of prairie dog colonies by ranchers and farmers caused the ferret population to decline. In 1973, the ferret became one of the first species to be listed as protected under the new US 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 came out to find out where the strange noises heard at night were coming from. He suspected that the Shep family's dog had gotten into a fight with some kind of reptile. Lying on the ground was a dead, tube-shaped, strange animal that the local taxidermist identified as a black-footed ferret. After biologists surveyed the area around Hogg's ranch, they were overjoyed to find a large colony of over 100 ferrets.
But after a few years, that colony also ran into trouble, shrinking to just a few dozen animals. In 1985, officials made the decision to round up all the ferrets they could find in hopes of starting a captive breeding program. They captured 18 individuals, but only seven survived to breed, putting the species at risk of inbreeding that could undermine the species' reproductive capacity.
Our heroine, Elizabeth Ann, owes her existence to a chance meeting at a banquet in Montana. In 1987, biologist Oliver Ryder studied the possibility of freezing animal cells. As a young scientist in the 1970s, he joined a new initiative known as the San Diego Frozen Zoo, which aimed to preserve the genetic material of a wide range of endangered mammals by freezing it in liquid nitrogen.
At an environmental conference, Ryder struck up a conversation with a Wyoming veterinarian named Tom Thorne, who told him about the plight of black-footed ferrets. A couple of years ago, Thorne sent several black-footed ferret specimens to the Frozen Zoo, but the scientists managed to save the cell line of only one: a male marked Studbook #2. Ryder suggested Thorn send more. Seven months later, he received skin cells from a female named Willa.
While this genetic material from the ghosts was in the freezer, the ferret breeding program, which has been run by the US Fish and Wildlife Service (FWS) since 1996, began to gain momentum. To date, about 10,000 ferrets have been born and many have been released into the wild. The program has been an outstanding success, but the ferret's survival is still far from assured.
One of the threats is the forest plague, a deadly bacterial disease introduced from Asia. Inbreeding also began to affect the population, because all the descendants descended from only seven animals. Genetic studies, Novak says, show they are all very closely related. The middle relationship is between brother, sister and cousin. As a result, potentially dangerous mutations have accumulated, says genome researcher Klaus-Peter Kopfli of the Smithsonian's Mason Conservation School. Some animals are born with curled tails and deformed chests.
To preserve the genetic diversity of ferrets, researchers have turned ferret breeding into a rigorous science. A computer program helps them assign an attractiveness rating to each possible couple based on the animals' origins. Breeders also relied on artificial insemination with genetic material taken in the 1990s from two prominent males known as Scarface and Rocky. But there isn't much material like this, says FWS's Pete Gobert, who coordinates recovery programs.
So, in 2013, program leaders approached Revive & Restore to see if they could support population diversity by turning small vials of “canned” frozen cages into living, breathing ferrets. We wanted to increase and maintain as much genetic diversity as possible from the small amount we had to start with,” says Gobert.
The idea was to use a technology that didn't exist when the zoo kept the specimens - somatic cell nuclear transfer. In this cloning technology, experts replace the nucleus of an egg with a nucleus taken from a cell in the body. An electrical discharge induces the egg and nucleus to merge, and the cell to reproduce. The embryo is then transferred to a surrogate mother. In 1995, Dolly the sheep became the first cloned animal. Since then, cloning has become a common breeding tool for farm animals, domestic animals, including cats and dogs, and even valuable racing camels.
Cloning to preserve the species has a much more patchy history. For example, when black-footed ferret breeders first turned to Revive & Restore, clones of just three endangered species were created in 2003, ungulates only: gaur (Bos gaurus) and European mouflon (Ovis aries musimon) in 2001 and banteng (Bos javanicus). In 2015, scientists cloned a fourth endangered species, a sheep known as the Isfahan mouflon (O. gmelini isphahanica). All died quite young (the banteng lived the longest, dying from injuries at the age of 7) and did not produce offspring. In part, cloning endangered animals has proven to be more difficult than cloning livestock or pets, as methods for breeding and keeping these species are less developed. Conservation programs also have fewer resources than commercial enterprises, so they are less likely to repeat a procedure after a failed attempt.
Cloning endangered species also faces ethical challenges. One is whether the clone, which may contain traces of its surrogate mother's DNA, really matches the species the researchers are trying to save. For example, clones of black-footed ferrets are created using cells from domestic ferrets, which means they carry the species' mitochondrial DNA, which remains in the cell after its nucleus is extracted.
Some conservationists have other concerns. They fear that the possibility of cloning rare species could undermine support for efforts to protect habitats and conserve species in the wild. And cloning can be costly, potentially diverting funds from other conservation efforts.
“I think cloning certainly has a future for endangered species, but there are some challenges,” says Barbara Durrant, director of reproductive science at the San Diego Zoos Wildlife Alliance. For each species, we need to be very careful when doing basic research,” she adds.
A few years ago, after rigorous technical and ethical reviews, federal regulators decided that the potential benefits of cloning ferrets outweighed the risks. Then, after obtaining permits, Revive & Restore teamed up with firms including pet cloning company ViaGen Pets and a commercial ferret breeder to develop a plan that cost about $40,000 to implement. It aimed to create embryos with DNA taken from Willa, a female black-footed ferret who died in 1988. Even ViaGen Pets lead scientist Sean Walker wasn't sure it would work. We knew we had checked all the points,” he says. But until you hear that heartbeat, you'll always be a little skeptical because there was a first time for everything.
In late 2020, the team implanted Willa's embryos into three pet ferrets and shipped them to the National Black-footed Ferret Conservation Center in Colorado, where about two-thirds of the population lives in captivity. To their relief, on December 10, 2020, one female gave birth to Elizabeth Ann. It was very exciting,” says Robin Bortner, a captive breeder.
But success was still not guaranteed. Two other pregnancies ended in failure and Elizabeth Ann had a stillborn littermate. And when Elizabeth Ann was placed with a second surrogate mother and her domesticated siblings, she often found herself in a dangerous place: at the bottom of a pile of puppies. For the first couple of days, we followed her very closely,” recalls Bortner. But as soon as Elizabeth Ann's eyes were opened about a month later, the audacity of a black-footed ferret awoke in her. Since then, Bortner says, she's been doing well.
Now fully grown, Elizabeth Ann looks and sounds like any other black-footed ferret. She eats meat and kills live hamsters. The caretakers don't want to risk giving her a live prairie dog that weighs more than she does. She yells angrily at people who get too close. She likes to attack and shred paper bags. And aside from her mitochondrial DNA, most of which is inherited from her domesticated mother, genetic analysis shows that she is 100% black-footed ferret.
This spring, scientists hope to conduct the mating process for Elizabeth Ann. Any offspring will still have Elizabeth Ann's mitochondrial DNA with traces of a domestic ferret. To remove these traces, any male offspring will mate with females in captivity, producing kits that no longer carry the domestic female's mitochondrial DNA. Successfully adding Willa's genes to the black-footed ferret gene pool through Elizabeth Ann would likely deal a blow to biodiversity, Novak says. Genomic analysis has shown that Willa's DNA contains 10 times more unique alleles than the DNA of any captive-bred ferret. This means that her chromosomes will provide an entirely new combination of traits and a higher level of genetic variants, says Kopfli. This should slow down the ferret's reproductive decline.
To bring even more genetic variation to captive-bred ferrets, ViaGen Pets plans to create a company for Elizabeth Ann. The scientists are improving the cloning procedure to be more efficient and will try to create the next batch of cloned ferrets in the spring of 2023 to coincide with next year's breeding season.
The successful use of cloning to save ferrets is likely to attract attention and possibly funding for similar efforts for other endangered species. But it won't be easy to repeat. This is partly because the species that can benefit from cloning must meet numerous criteria. For example, the best candidates have ready-made genetic material and a less endangered close relative who can act as a surrogate mother. It also helps to get funding and infrastructure for captive breeding. Few programs yet can meet these requirements.
Another hurdle is that while the basic cloning process is the same for all mammals, the technology works better in some species than in others, and no one knows why, says Durrant. To increase the chances of success, researchers often have to develop a unique "recipe" that takes into account the animal's reproductive characteristics, says Samantha Weisley, a conservationist at the University of Florida who works on the Black-footed Ferret program. Reproductive technology is specific to different species.
However, efforts are ongoing to clone at least two other endangered species. One of them is the Przewalski's horse, a stocky wild horse that once roamed Europe and Asia. This species almost became extinct in the middle of the 20th century, and all individuals living today are descended from only 12 animals. Fortunately, about 300 cell variants have been preserved in the Frozen Zoo, and now conservationists are trying to bring some of this lost genetic diversity into modern populations. In 2020, researchers created Kurt, a clone of Przewalski's horse whose cells were frozen 40 years ago. Although he was born a few months before Elizabeth Ann, he still needs to grow up a bit before he is ready to breed. By spring 2023, cloned siblings may join Kurt.
The San Diego Zoo Wildlife Alliance, a non-profit organization, is also in the preliminary stages of trying to clone the northern white rhino (Ceratotherium simumcottoni), the most endangered of several rhino subspecies. Only two northern white rhinoceros are left alive, and none of them is capable of having offspring. As a first step, the scientists are working on methods for incorporating the genome of the northern white rhino into cells from a close relative, the southern white rhino (C. simum simum). And once the team refines their methods, we certainly hope to be able to apply them to black rhino, Sumatran rhinoceros and possibly Javan rhinoceros,” says Durrant.
Some researchers are considering not only cloning, but also other genetic technologies that could help endangered species. Birds, for example, cannot be cloned, but Revive & Restore recently formed a research consortium to develop a technique that could do the same. It involves the introduction of primary germ cells of an endangered species into an embryo of a surrogate species, such as a chicken. These germ cells then migrate to the gonads of the chicken. Thus, a domestic rooster may subsequently become the parent of, say, the endangered prairie chicken.
CRISPR gene editing tools may also play a role in conservation. For example, editing the genome of black-footed ferrets so they can withstand wild plague could be a game-changer. Kopfli and his collaborators are now comparing the genome of the black-footed ferret with that of its domestic relative, which is not susceptible to the disease, in the hope of determining the genetic basis for resistance. But in reality, genetically modifying ferrets and then releasing them into the wild would require extensive legal and ethical discussion.
At the moment, Elizabeth Ann's guardians are just getting ready to choose her ideal companion. Every male ferret in six kennels scattered across the United States and Canada undergoes a thorough inspection. This is partly because Elizabeth Ann will likely only have a few good breeding years, and annual litters average three to five cubs. that an aggressive ferret would harm their only creation.
This month they will shortlist and make their choice. If the most suitable option turns out to be in another country, it will be sent to Colorado. All necessary samples will be taken in case they are needed for the artificial insemination of Elizabeth Ann. But everyone hopes that once two ferrets meet, nature will just take care of itself.