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For over a decade, scientists have pursued the concept of de-extinction, aiming to revive extinct species. Now, Colossal Biosciences has seemingly achieved a significant step in this endeavor with the dire wolf, a massive, prehistoric species popularized by the TV series “Game of Thrones.”
Reviving the Dire Wolf: A Step Closer to De-extinction
In 2021, a separate group of researchers successfully extracted DNA from dire wolf fossils, creatures that vanished approximately 13,000 years ago. Building upon this discovery of further genetic material, Colossal Biosciences scientists have now genetically modified 20 genes in gray wolves to incorporate essential dire wolf characteristics. Subsequently, they generated embryos from these altered gray wolf cells and implanted them into surrogate dog mothers, awaiting birth.
The Birth of Pups with Dire Wolf Traits
The outcome is the birth of three healthy pups – two males, six months old, and one female, two months old, named Romulus, Remus, and Khaleesi. These canines exhibit certain dire wolf attributes.
- Larger Size: They are notably bigger than typical gray wolves.
- Distinct Coat: They possess dense, pale fur, unlike the coats of gray wolves.
Colossal Biosciences, recently valued at $10 billion, houses these pups at a secluded 2,000-acre facility in an undisclosed location in the northern United States.
“Functional Copies” of an Extinct Species
Beth Shapiro, Chief Scientific Officer at Colossal Biosciences, described the wolf pups as the inaugural successful instance of de-extinction. “We are producing functional replicas of something that once lived,” she stated in an interview.
While these animals will remain in controlled environments, the technology developed by Colossal Biosciences holds promise for conserving extant species, such as the critically imperiled red wolf, primarily found in North Carolina.
In 2022, red wolf-coyote hybrids were identified in Texas and Louisiana. Colossal Biosciences also announced the creation of four clones from these hybrids, suggesting that introducing these clones into North Carolina could potentially enhance the genetic diversity of the red wolf population and aid in preventing their extinction.
Approaches to Species Revival
Over time, scientists have proposed various methods for resurrecting extinct species. For example, the hypothetical recovery of an intact cell from a frozen woolly mammoth carcass could potentially enable the creation of a mammoth clone upon thawing.
However, the founders and scientists of Colossal Biosciences, established in 2021, adopted a different strategy. They focus on analyzing ancient DNA to pinpoint key genetic variations that distinguished extinct species from their living relatives. Researchers then genetically engineer the DNA of a living relative, employing these genes to produce viable animals. These revived animals would not be genetically identical to the extinct species but share crucial characteristics.
Focusing on the Dire Wolf: Overcoming De-extinction Hurdles
Initially, Colossal Biosciences launched ambitious projects targeting woolly mammoths and the dodo, a flightless bird extinct for three centuries. Nevertheless, challenges soon arose.
Technical Difficulties in Genetic Modification and Reproduction
One significant hurdle was the complexity of making numerous genetic edits. While single gene edits are relatively straightforward, the aim was to make dozens. Furthermore, generating animals from the modified DNA presented difficulties. The initial vision of growing mammoth fetuses in Asian elephant surrogates lacked prior precedent in elephant in vitro fertilization. Similarly, resurrecting a dodo required inserting a modified bird embryo into a hard-shelled egg.
In 2023, the Colossal Biosciences team shifted their focus to dire wolves, considered a more attainable initial objective. Given the close relation of dire wolves to domestic dogs, scientists could capitalize on existing research in dog cloning and embryo implantation.
“We have conducted extensive research on dogs, stemming from the public’s affection for the domesticated gray wolf,” Dr. Shapiro explained.
Unlocking Dire Wolf Genetics
Dr. Shapiro, joining Colossal Biosciences in 2024, was involved in the 2021 team that first extracted dire wolf DNA from fossils. However, this initial effort yielded limited genetic data. At Colossal Biosciences, she and her colleagues pursued further dire wolf DNA, aiming to enhance understanding of the extinct species’ biology and potentially facilitate its revival.
“It was the most direct route to achieve a predictable outcome,” Dr. Shapiro noted.
The research team re-examined dire wolf fossils, employing advanced DNA isolation techniques. This time, they successfully obtained a substantial amount of genetic material from two fossils: a 13,000-year-old tooth from Ohio and a 72,000-year-old skull from Idaho. The recovered dire wolf genomes enabled Dr. Shapiro and her team to reconstruct the evolutionary history of dire wolves in greater detail.
Analysis revealed that dire wolves belonged to the same evolutionary branch that gave rise to modern wolves, jackals, and African wild dogs. The dire wolf lineage diverged approximately 4.5 million years ago. Subsequently, around 2.6 million years ago, dire wolves interbred with other species, including ancestors of contemporary gray wolves and coyotes.
According to Julie Meachen, a paleontologist at Des Moines University involved in the ancient DNA project, dire wolves were dominant across southern Canada and the United States. They outcompeted gray wolves, possessing 25 percent larger size and stronger jaws and teeth. Their prey included horses, bison, and potentially mammoths. The extinction of many of these prey species, possibly exacerbated by human hunting, may have sealed the dire wolf’s fate, allowing gray wolves to expand from northern Canada and Alaska and occupy the ecological niche.
Dr. Meachen and her colleagues discovered that dire wolves and gray wolves share over 99 percent genetic similarity. Eighty genes displayed significant differences, some known to influence size in extant dogs and wolves, suggesting their role in the larger size of dire wolves.
Further surprising was the finding that dire wolves carried genes for a light-colored, likely thick and dense coat. Dr. Shapiro and her team are preparing a publication detailing these findings.
Blueprint for a Dire Wolf
Equipped with a comprehensive list of dire wolf genes, the Colossal Biosciences scientists initiated their de-extinction project.
Initially, they extracted cells from gray wolf blood and cultured them in vitro, subsequently manipulating the wolf DNA.
A decade prior, researchers had modified a single gene in beagles to induce increased muscle mass. Since then, techniques for editing multiple genes simultaneously in mammalian DNA have advanced. For the dire wolf project, the Colossal Biosciences team aimed to edit 20 genes, pushing the boundaries of current technology.
Scientists introduced dire wolf mutations into 15 genes, omitting five others due to prior research indicating those mutations cause deafness and blindness in gray wolves.
Instead, the Colossal Biosciences team identified alternative mutations for those five genes present in healthy dogs and gray wolves. They incorporated these alternative modifications into the gray wolf cells.
“It’s a delicate balance,” Dr. Shapiro explained. “The objective is to resurrect these traits, without causing harm to the animal.”
The researchers then transferred the modified DNA from the gray wolf blood cells into enucleated dog eggs. They generated numerous such eggs, implanting them into large dogs as surrogate mothers.
While most embryos failed to develop, four pups were born. One pup died after 10 days due to a ruptured intestine, but autopsy results indicated the death was not linked to harmful genetic modifications.
Matt James, Chief Animal Officer at Colossal Biosciences, oversaw the pregnancies and births. He recognized the experiment’s success immediately upon seeing the white coat of a pup.
“That initial glimpse of white was truly striking,” Dr. James recalled. “It’s an image that will forever be etched in my memory.”
Two pups, Romulus and Remus, are named after the legendary founders of Rome, raised by a wolf in myth. The third pup, Khaleesi, is named after a prominent character from “Game of Thrones.”
Dr. James noted the pups are approximately 20 percent larger than gray wolves of the same age. In addition to their white, thick fur, they also exhibit exceptionally bushy tails and a mane-like ruff around their necks.
Researchers are monitoring the wolves’ growth and observing for any unforeseen biological changes. “I am intensely curious to see what develops,” Dr. Shapiro commented.
She added that the captive rearing of these animals would likely limit insights into natural dire wolf behavior.
“I would be fascinated to understand the natural behavior of a dire wolf,” she stated. “However, they are essentially experiencing a luxurious, protected existence. We are aware of even the smallest issues they might encounter.”
Adam Boyko, a geneticist at Cornell University, not involved in the project, acknowledged that “it’s exciting that we can create functional representations of extinct species.” However, he questioned whether Romulus, Remus, and Khaleesi are genuine resurrected dire wolves. He pointed out they are not raised in dire wolf packs to learn species-specific behaviors, nor do they consume an ancient diet to acquire their ancestors’ unique gut microbiota.
While these animals carry 20 dire wolf genes, potentially revealing aspects of the extinct species’ biology, Dr. Boyko speculated that numerous other genes also contributed to their distinctiveness from other wolves. “The number of genes involved remains unknown,” he said. “It could be 20, or potentially 2,000.”
Colossal Biosciences is collaborating with several Native American communities in the United States. The MHA Nation in North Dakota has expressed interest in the dire wolf project. “Their presence would remind us of our responsibility as guardians of the Earth,” Mark Fox, MHA Nation tribal chairman, said in a company statement.
However, introducing animals with dire wolf DNA into the wild would require them to survive in a drastically altered environment compared to the Ice Age. The large prey species dire wolves specialized in hunting are largely extinct or exist in diminished populations. Any reintroduced, free-roaming dire wolves would need to prey on smaller animals and potentially compete with gray wolves.
Furthermore, gray wolves and red wolves currently face threats, including hunting, that genetic manipulation cannot address.
Last month, 60 environmental organizations protested a congressional bill aimed at removing gray wolves from the endangered species list, a change they warn could increase hunting-related deaths.
“If enacted, this bill would effectively ensure the demise of thousands of wolves nationwide,” they wrote.
Dr. Meachen, uninvolved in the pup creation, expressed ambivalent feelings regarding the de-extinction endeavor.
“Part of me, the child within, is excited to see what they look like,” she admitted. “But I also have concerns. We are already struggling with the wolves we have now.”