Some of the mummies that have been recovered from the Siberian permafrost are impeccably preserved, with intact tissues, hair, and internal organs that are clearly visible in CAT scans and autopsies. Oddly, the DNA within even the best preserved mummies tends to be in bad shape compared with the DNA that is preserved in bones. One possible explanation is the difference in the amount of time it takes to freeze the DNA. If body parts are scavenged and the flesh consumed by predators, the de-fleshed bones are likely to be rapidly buried and frozen in permafrost, while mummies would stay warm for far longer. While the mummy was slowly freezing, microbes from the animal’s gut and the environment would colonize tissues throughout the body, decomposing the animal from the inside and simultaneously destroying the DNA.
Although the record of DNA preservation in mummies is startlingly poor, we can’t seem to separate the remarkable physical preservation of their bodies from the idea that their DNA must, somehow, be equally well preserved. With each find, there is renewed enthusiasm that this mummy will be the one that defies the odds. This is the mummy that will have intact cells with intact nuclei that contain intact genomes. This mummy will have the donor cells for cloning by nuclear transfer.
The first I heard of Bernard Buigues was just after one of these remarkable finds. It was October 1999, and a mammoth that no doubt had intact cells and intact nuclei with intact genomes had just been flown across the Siberian tundra.
Whenever there is a spectacular result in the ancient DNA world, my colleagues and I are inundated with calls from journalists looking to be the first to break the story about the imminent resurrection of the mammoth/dinosaur/dodo. On this particular day, I was sitting at my desk in Alan Cooper’s ancient DNA lab at the University of Oxford. It was my first month as a PhD student and immigrant to the United Kingdom.
The phone rang, and I answered it. The caller launched into a series of rapid-fire questions, speaking in an accent that was unfamiliar to my American ears. I made out the words “helicopter” “jackhammer” “cryogenics” “tusk” and “Siberia” but did not manage to find a break into which insert a response (such as “Could you please call back when someone who’s been at this for more than two weeks is in?”). The journalist then paused and, much more clearly, asked me what my opinion was about whether a hair dryer could ruin the chances of cloning a mammoth.
I was pretty sure I could have an opinion about a hair dryer and its role in cloning a mammoth. I was also sure that, since I wanted eventually to be taken seriously as an ancient DNA scientist, I should probably ask for clarification before offering an opinion.
I learned that a team of arctic explorers led by my soon-to-be friend and colleague Bernard Buigues had just unearthed what they believed to be a nearly complete mammoth mummy. In a drastic and dramatic attempt to keep the mammoth cells frozen and therefore intact, they left the slightly decaying corpse in the ground until winter so that the ground was good and frozen. Then, using jackhammers and strong shovels and working in the freezing dark, they cut a 21,000 kilogram block of frozen dirt out of the permafrost and flew it, hanging off the underside of a large helicopter, nearly three hundred kilometers back to Bernard’s underground cave in Khatanga, where they planned to slowly and methodically thaw the mammoth carcass out of the ice using a hair dryer.
For good measure, and because it made the pictures and video even more impressive, Bernard (who admits that he was using “creative license” when he did this) stuck the tusks that had been found near the exposed skull into the side of the frozen block of ice before the helicopter took off, so that it looked like there was a complete mammoth inside a frozen box flying across the tundra. They knew that the mammoth carcass in the block of ice was incomplete. They had already removed the head, for example, which had partially thawed and begun to rot. They had also used ground-penetrating radar to try to see beneath the surface, and the results hinted that less than a complete mammoth was preserved within. But they were hopeful.
This mammoth, which was named Jarkov after the local family who discovered it, lived around 23,000 years ago. Jarkov was an adult male mammoth, about three meters tall, that probably died a few years before his fiftieth birthday. The idea that Jarkov could be cloned was floated almost immediately. This idea was embraced in particular by the Discovery Channel, which funded Jarkov’s dramatic extraction from the ground. Larry Agenbroad, a mammoth expert from Northern Arizona University, reported in the team’s press release that they had already lined up a lab with expertise in cryogenics and “elephants available.”
A year later, the hair-dryer defrost revealed only a small amount of mammoth preserved within the giant block of dirt. Even more disappointingly, what was preserved was mostly bone, with a bit of tissue and some hair. No intact nuclei were discovered, but short fragments of DNA extracted from the hair were used to construct a complete mitochondrial genome and, eventually, part of the mammoth nuclear genome. Jarkov would not be the first cloned mammoth. However, the spectacle of his extraction from the earth and flight across the tundra instilled in the public a sense of just how important a frozen mammoth would be for mammoth cloning. It also reinforced the (incorrect) assumption that what we really needed to find was a whole, perfect mummy.
One year before the spectacle of the Jarkov mammoth flying across the tundra, a team of Japanese scientists led by Akira Iritani and Kazufumi Goto founded the Mammoth Creation Project, whose goal was clearly stated in the name. Iritani and Goto were involved with in vitro fertilization research in Japan, and both had made fascinating discoveries about the hardiness of sperm. For example, they learned that sperm taken from cows and pigs and frozen to –20˚C could be defrosted and used to fertilize eggs, from which perfectly healthy cows and pigs would develop. Having read about Zimov’s Pleistocene Park, they wondered whether frozen mammoth sperm might be key to resurrecting the park’s star attraction.
With mammoth sperm on his mind, Iritani set off on a series of Siberian expeditions in search of a frozen bull mammoth. The expeditions were led by Petr Lazarev, a geologist and head of the Mammoth Museum in Yakutsk. If they were successful in finding bull mammoths, Iritani and Goto planned to harvest their sperm and use it to fertilize the eggs of elephants. Because this would result in a hybrid calf and not a cloned mammoth, they intended to use sperm that contained the X chromosome and make only females. Then, when the hybrid females became sexually mature, they would impregnate her with embryos created using her eggs and other mammoth sperm. In this way, Iritani predicted that he would be able to create a creature whose genome would be 88 percent mammoth within only fifty years.
After two summer expeditions in 1997 and 1998, the Mammoth Creation Project had no money left and no mammoth sperm to show for their effort.
Then, in 2002, the Yukagir mammoth was discovered.
A FIRST ATTEMPT
In the autumn of 2002, Vasily Ghorokov was out hunting for tusks along the banks of the Maxunuohka River in Yakutia, northern Siberia. Ghorokov and his sons spotted the tip of what looked like a particularly well-preserved specimen and began to dig. As he reached the base of the tusk, he realized that it was still attached to what turned out to be most of a skull, which was so well preserved that parts of it were covered in skin and hair. Word spread quickly about the new find, and competing groups hurried to find a way to reach the site. Buigues learned of the find via his extensive connections across Siberia. In Yakutsk, the news reached Lazarev at the Mammoth Museum. Lazarev called Iritani and revealed plans to continue the excavation the following autumn. And Iritani decided that at seventy-one, he was too old for yet another Siberian expedition. He would instead send one of his students.
A year later, a team of international scientists arrived at the site of the Yukagir mammoth. Buigues led the team, which included, among others, Iritani’s student Hiromi Kato, Petr Lazarev, and Alexei Tikhonov, the scientific secretary of the Russian Mammoth Committee who was based at the Zoological Institute in Saint Petersburg. In this second season
of excavation, the team painstakingly recovered the mammoth’s left front leg, taking extreme caution to keep it frozen. Like the skull, the leg was impeccably preserved and covered in soft tissue and hair.
Then the trouble began. A rival Japanese team surfaced and offered a hefty reward to anyone who could provide a mammoth that could be a main attraction at the upcoming 2005 World Expo. Export permissions became impossible to obtain. In the end, the leg had to stay. Kato returned to Iritani empty-handed, no closer to a cloned mammoth. Lazarev, doing his part, snagged a bit of the foreleg tissue and carried it personally to Iritani in Japan, but by the time he arrived the flesh had begun to decay.
After one more autumn excavation, this time directed by Naoki Suzuki of Jikei University in Tokyo, the Yukagir mammoth was removed entirely from its tundra grave. Parts of the vertebral column and rib cage were recovered, as was a portion of intestine packed with fecal material. Scientific analysis of these remains revealed that when the Yukagir mammoth died around 22,500 years ago, it was forty-eight years old and weighed somewhere in the range of 3,500 to 4,500 kilograms, which was average for an adult male mammoth. Suzuki would eventually oversee transport of the Yukagir mammoth to Japan, where it would be intensively studied using X-ray computer tomography, providing the first internal anatomical scan of a mammoth without causing any harm to the specimen. While in Japan, the Yukagir mammoth was a centerpiece at the 2005 World Expo in Aichi.
After its stint in Japan, the Yukagir mammoth was flown back to Yakutsk, where it is currently stored in an underground cave in the center of the city, where frozen fish and reindeer and other food is stored (plate 14). A few summers ago, I had an opportunity to see the Yukagir mammoth myself. It sits in the far back corner of the cave, in a compartment of its own. The Yukagir mammoth is as impressive as the hype suggests. However, no intact mammoth cells have been recovered from its body, despite its extremely good state of preservation.
A few years ago, Iritani and his team published a research paper in the Proceedings of the Japan Academy, in which they described the first experiment to clone a mammoth using nuclear transfer. Iritani’s team extracted cells from the bit of foreleg that Lazarev managed to get out of Russia, including cells from what appeared to be preserved bone marrow. Iritani’s team prepared mouse eggs for nuclear transfer by removing the mouse nuclei. They inserted nuclei that they managed to extract from the Yukagir mammoth’s cells into the prepared mouse eggs. If the genomes within these mammoth cells were sufficiently intact, the mouse egg would hopefully trigger the mammoth somatic cells to de-differentiate into stem cells, and development would begin.
Nothing happened.
A BETTER MAMMOTH AND A POSSIBLE SOLUTION TO THE PRESERVATION PUZZLE
In 2007, the three sons of Yuri Khundi, a Nenet reindeer herder, discovered a nearly perfectly preserved baby mammoth along the banks of the Yuribey River in northeastern Siberia. Khundi wanted the mammoth, but he wasn’t sure how to get it out of the tundra. The Nenets believe that mammoths are bad luck—beasts that wander the darkness of the frozen underworld. Electing not to risk the retribution of the beasts, Khundi and a friend decided to see whether the director of a local museum had any ideas. Sensing something important, the museum director convinced the local authorities that they should help. The entire crew then went back to the Yuribey River. When they got there, there was no baby mammoth.
It turned out that one of Khundi’s cousins had heard the story of the baby mammoth by the river and, less concerned with bad luck than with good fortune, decided to go get it himself. Khundi was not happy about the turn of events. He found out that his cousin had been seen heading for a nearby town, so Khundi and his friend followed. When they arrived, they found the mammoth propped against the wall of a store and looking a little bit worse for wear. Khundi’s cousin had sold the mammoth to the store’s owner in exchange for a year’s worth of food and two snowmobiles. Unfortunately for the mammoth, local dogs had been chewing bits off of its extremities whenever the store owner’s back was turned.
The story has a happy ending: Khundi managed to reclaim the baby mammoth before much more damage was done, and the mammoth was moved to the Shemanovsky Museum in Salekhard for safekeeping.
The mammoth, a female that was later named Lyuba, was only a month old when she died 42,000 year ago. She was so well preserved that her stomach still contained traces of her mother’s milk. About a year after her discovery, researchers including Bernard Buigues, Dan Fisher, Alexei Tikhonov, and Naoki Suzuki performed a marathon three-day autopsy of Lyuba’s body in a lab in Saint Petersburg, Russia. They discovered fine mud in her lungs, mouth, and throat, which likely meant she had died of asphyxiation, perhaps while trying to cross a muddy river. They studied her baby tusks, looked for mites in her hair, and learned that, like elephants, mammoth babies ingest their mother’s feces to inoculate their digestive systems with the microbes that will break down the plants they eat. And, in an important step for anyone interested in cloning a mammoth, they discovered why Lyuba was so well preserved.
Dan Fisher, one of the members of our expedition team during that unproductive summer on the Taimyr, was key to solving this puzzle. Dan is a soft-spoken man who knows a lot about mammoths. His interest in mammoths, however, is not limited to the animals themselves. He also cares a great deal about how people interacted with mammoths. For example, mammoths were certainly too big to eat in one sitting. One question that Dan seeks to answer is, how did mammoth hunters preserve meat in the absence of modern refrigeration?
While we were in the field, Dan told us about a series of experiments that he had performed near his home in Michigan to see how long meat would remain edible if it were stored in shallow ponds. First, he butchered lamb and venison and anchored the meat to the bottom of shallow ponds in a nature reserve associated with his university. Over a period of two years, he would bring up the meat every now and then and check for decomposition. Then, one day in mid-February of 1993, a colleague gave him a draft horse that had just died of natural causes. This gave Dan a new idea. Using stone tools that he fashioned himself, mimicking as best he could the technology of the mammoth-hunting indigenous people from the Great Lakes Region, he butchered the horse. It was winter and the ponds were covered in ice. So he chopped a hole through the ice and submerged the horse meat in the cold water. Every two weeks, he brought the meat out and cut off a piece to test for palatability and signs of decay. By June, Dan noted that the meat, while still retaining considerable nutritive value, had developed a sour taste and a strong, sour odor. This was the same strong, sour odor that Dan noticed coming from Lyuba’s carcass as they performed their autopsy in Saint Petersburg.
The sour odor was caused by microbes called lactobacilli. Lactobacilli convert lactose and other sugars to lactic acid and are found naturally in the guts of many animals. The buildup of lactic acid in Lyuba had effectively pickled her, preserving her in the permafrost where she was buried and protecting her from decay even after her body was exposed.
Unfortunately, although high acidity might be good for pickling mummies, it is not good for DNA preservation. These mummies may appear to be very well preserved, but the high-acid environments cause considerable cellular damage and destroy naked DNA. That means that, while these mummies might appear—superficially—to be the most likely source of an intact cell suitable for cloning, their remains may actually be the worst place to look for such cells.
Some scientists remain undeterred, however, and the race to clone a mammoth remains in full force. Teams are still out every summer looking for mammoth mummies, hoping that one day an exceptionally preserved mummy will emerge, unpickled, from the Siberian tundra.
UPPED STAKES AND A NEW CONTESTANT
In 2008, Teruhiko Wakayama of the Tiken Centre for Developmental Biology in Kobe, Japan, cloned mice that been frozen at–20˚C for sixteen years. This was a huge and important step for the de-extinction effort, for two reasons. First, all of the cells that Wakayama and his team use
d were dead before they were injected into prepared mouse eggs. That meant that mammoth hunters might not need to find a living cell in order for nuclear transfer to work, because, sometimes, even dead cells contain sufficiently intact genomes for cloning. Second, they discovered that they could increase the chances of success of nuclear transfer by adding a step to the cloning protocol. Their results suggested that some cells, particularly those whose genome might be a little bit broken, may simply need an extra push in order to be fully de-differentiated.
Initially, Wakayama’s team followed the standard protocol of nuclear transfer: isolating nuclei from the once-frozen mouse cells and inserting them into prepared mouse eggs. Although not many of the eggs began to develop, a few did, indicating that the egg was able to reset some of the somatic cells. However, none of these went on to become fully developed mice. Instead, the process stalled after a few cell divisions, suggesting that de-differentiation was not completely successful.
Then they had an idea. They repeated the process, but this time they stopped the embryo from developing after only a few rounds of cell division. They then took those cells that had started to develop and used them to create what are called cell lines—large colonies of identical cells growing in the lab. Next, they removed nuclei from these growing cells and inserted them into a freshly prepared egg. In this way, the egg had not one but two chances to reprogram these cells into completely differentiated stem cells. To the astonishment of the scientific community, two of the embryos created in this way went on to develop into healthy, adult mice.
How to Clone a Mammoth Page 10