The holidays came early for conservation biologists in 2020 when the U.S. Fish and Wildlife Service announced on 10 December that scientists had successfully cloned a wild black-footed ferret—using DNA from a ferret that died years earlier. Photographs flashed around the world, showing the slender, playful, khaki-colored kit.
She was the first native endangered species to be cloned in the United States. The scientists named her Elizabeth Ann.
Black-footed ferrets are so uncommon that in the 1970s they were written off as presumptively extinct. The discovery of a colony of black-footed ferrets in Meeteetse, Wyo., in 1981 led the U.S. Fish and Wildlife Service to embark on managed breeding programs and reintroduction. Some of the wild ferrets were infected with canine distemper or sylvatic plague and died before they reproduced. At one point, scientists had only seven black-footed ferrets with which they set out to rescue the species.
Their managed-breeding program has succeeded in producing in excess of 10,000 individuals, 400 to 500 of which have been reintroduced to the wild. However, that achievement is tempered: every black-footed ferret today is closely related to every other black-footed ferret, and they all have uncomfortably similar genomes. This reality—common among managed breeding programs that begin with few progenitors—puts the species at risk of inbreeding and makes it more susceptible to the rapid spread of disease.
Solving that problem requires introducing new genetic variation into the population. But where does one find new genetic material when a species is down to its last seven living individuals? The answer: non-living individuals. That is, the DNA preserved in frozen tissue samples of black-footed ferrets that Dr. Kurt Benirschke, a German-American pathologist and geneticist, had the vision to collect and store decades earlier. As founder of the San Diego Zoo Wildlife Alliance’s research department, Benirschke in the mid-1970s started collecting tissue samples for preservation in the San Diego Wildlife Alliance Frozen Zoo® that could, he surmised, someday be used to clone extinct animals—an idea not universally accepted at the time because much of the necessary technology had yet to be invented.
But Benirschke, who died in 2018, and his successor, Dr. Oliver Ryder, are now seen by many as leaders in seeking ways to use genomic tools to pull threatened and endangered species back from the brink of extinction. The Frozen Zoo, a biobank within the Beckman Center for Conservation Research—one of more than 340 such cryogenic repositories around the world—now holds 10,200 cell cultures from more than 1,200 species and subspecies. There is also frozen sperm from more than 400 species. All of the holdings are duplicated offsite, for safety.
“People realize—wow—none of the discoveries we are seeing today would have been possible without creating that biobank,” said Klaus Koepfli, a senior research scientist in the Smithsonian-Mason School of Conservation.
Ryder is proud of the Frozen Zoo, but clear-eyed that the facility’s goal is not only to collect and preserve genetic material but to save waning species. “The question is,” he said of the 10,000 cell cultures, “can these be used for restoring genetic variation?”
He was talking about more than the black-footed ferret. The Zoo also has used tissue samples in its frozen zoo to clone a Przewalski’s horse, an animal given up as extinct in the wild in the mid 1990s. The clone, born in 2020 and named Kurt in Benirschke’s honor, was created by replacing the nucleus of a horse’s egg with that of a Przewalski’s horse, which has 66 chromosomes, two more than domestic horses, and implanted the egg in an American quarter horse to gestate. After Kurt was born, he was paired with another Przewalski’s horse, Holly, who is about Kurt’s age and was raised in a herd of their species. Romping with Holly will help Kurt “learn” to communicate with other Przewalski’s horses.
The biobank also helped Cynthia Steiner, a conservation biologist at the San Diego Zoo Wildlife Alliance in San Diego, Calif, discover why a disease called chondrodystrophy afflicted a small number of California condor eggs in the otherwise successful breeding program to rescue the bird. A review of all condor blood samples in the frozen zoo revealed that three of the 14 founders—the birds from which all others descended—carried the genetic mutation responsible for chondrodystrophy. Steiner and her colleagues keep the disease at bay by not pairing two carriers when breeding the birds.
Biobanked stem cells from northern white rhinos may be the majestic species’ last and best hope for survival. Only two of the rhinos exist, but reproductive and conservation biologists are working with academic researchers to use the stem cells to develop rhino sperm and oocytes. Biologists could then use them to develop artificial insemination, in vitro fertilization and embryo transfer skills.
The black-footed ferret project began in 2013, when the U.S. Fish and Wildlife Service approached Revive and Restore to learn how biotech might help the agency save endangered species from extinction, which is part of its legal mandate. Revive and Restore, a nonprofit organization that facilitates and sometimes pays for using biotechnology in wildlife conservation, reached out to the San Diego Zoo Wildlife Alliance, which had the tissue samples required for cloning, and ViaGen Pets and Equine, a private company in Texas, that routinely clones cattle, horses, even camels, as well as pets.
Even when the team was assembled, scientists spent several years conducting experiments to see what would happen when they inserted a black-footed ferret genome into a domestic ferret egg. Would the embryo develop and grow? Would such embryos gestate normally in a surrogate mother? Satisfied with the answers, the team implanted a clone and about 42 days later, on 10 December 2020, Elizabeth Ann was born in a U.S. Fish and Wildlife facility in Colorado.
“Thanks to Elizabeth Ann, we she showed the world that we can reach back 40 years into the freezer and produce an animal,” said Ben Novak, lead scientist of Revive and Restore.
That is true, but cloning was just the means to an end. Elizabeth Ann was created to introduce new genes into the black-footed ferret population, but she cannot. She is afflicted with hydrometra, a persistent accumulation of fluid in the uterus; the only permanent fix is to surgically remove the organ. She will never pass along the 40-year-old genes needed to help her species step back from the brink.
Ryder was disappointed by the news but undaunted. He kept reminding himself that the process had to be “stepwise and intentional.”
“First,” he said, “can we make a ferret with using cross species cloning and embryo transfer?” The photogenic ferret was evidence that they could, and had.
“Will clones behave normally like (naturally bred) ferrets?” was his next question. “Elizabeth Ann very much falls within all of the characteristics of black-footed ferrets. She is a rather ferocious, spicy little creature.” Indeed, before veterinarians diagnosed her hydrometra, the waspish Elizabeth Ann chased off four potential mates, biting one of them on the nose.
“But,” Ryder continued, “what will her offspring be like? Well, you know this: she won’t produce any offspring, but other clones will. And they will be judged. Do they have normal growth rates? Do they behave normally? Are they healthy?
“If they pass that test,” he concluded, “then there will be some effort to intentionally incorporate that genetic diversity to expand the gene pool.”
Budhan Pukazhenthi, chair of the AZA Biobanking Scientific Advisory Group, echoed that go-it-slow approach. “Cloning an offspring gets everyone excited,” said Pukazhenthi, a research physiologist at the Center for Species Survival in the Smithsonian’s Conservation Biology Institute in Front Royal, Va., “but there has been very similar tech in the past that hasn’t worked.”
Koepfli, among others, are eager to give this technology another chance. The potential benefits—saving one species and perhaps demonstrating how to save others—are too important to pass up.
“That (cloned) black-footed ferret may contain a number of genetic variants that haven't been in any of the black-footed ferrets bred since the late 1980s,” he said. “That’s a way to introduce that new genetic variation to the population.”
Mark Stein is a writer based in Larchmont, N.Y.
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