CREATE A CLONE
When you are working in the tundra, nobody cares if you sing loudly and out- of-tune as you walk along a meandering river. Nobody laughs at the five layers of clothing you’re wearing or mocks the variety of nets you’ve donned in your latest, ill-fated attempt to limit mosquito access to your flesh. And nobody bats an eye when your battered Mi-8 helicopter makes an unexpected stop in the middle of the Siberian tundra and picks up a French-speaking couple, their five-year-old child, and a large, red cooler.
These are lessons I learned in the summer of 2008, during what I fondly remember as my strangest and least successful bone-hunting season. That summer, we spent several weeks living in a small encampment surrounded by lakes on the low-lying tundra of the Taimyr Peninsula. We were hunting mammoths.
The Taimyr expedition was led by Bernard Buigues, a seasoned and pleasantly eccentric arctic explorer, and there was no reason to suspect it would not go well. For decades, Bernard, as president of CERPOLEX (CERcles POLaires EXpédition), led overland expeditions into Siberia and to the North Pole. These expeditions began from his well-appointed base in Khatanga, a small Russian town situated on the Khatanga River in Krasnoyarsk Krai. By the early 2000s, Bernard’s interests had shifted to expeditions of a more scientific variety, and he had formed Mammuthus, an organization attached to CERPOLEX with the stated goal of exploring and celebrating the Arctic and its many treasures. As the name implies, however, Mammuthus was particularly focused on recovering and facilitating scientific investigations of mummified remains of mammoths. The formation of Mammuthus was either opportunistic or timely, as mummies of mammoths and other ice age giants have been popping out of the Siberian permafrost at a surprising rate since the turn of this century.
Upon meeting Bernard, it was impossible not to have complete faith in both his leadership and the success of the expedition. By 2008, Bernard had decades of experience working in the Siberian tundra. He had seemingly endless energy and enthusiasm, a deep knowledge of the logistical challenges of working in Siberia (and how to circumvent these challenges), and a large collection of warm coats. Most importantly, he had a long history of collaboration with the people living in the region, which goes some way to explain why he is so often the first to get access to newly discovered mammoth mummies. By all reasoning, the expedition should have gone well.
It was at Bernard’s Siberian home in Khatanga where our adventure began. Khatanga is an unusual place. It is one of the most northerly inhabited places in the world. Although fewer than 3,500 people live there, it has an airport, a hotel, and a natural history museum full of artifacts relating to the region’s people and history. Khatanga also has a few restaurants serving locally sourced meats flavored with dill, and several small stores selling US$8 frostbitten carrots, semiautomatic machine guns, and a bizarre variety of flavored chewing gum. The roads and riverbanks are littered with unfamiliar machines, some of which possibly work. And the people live in anything from small wooden houses to large apartment buildings or even shipping containers—the kind that are stacked on container ships for transport across the ocean. Even Bernard’s house was partly made of shipping containers strung together and, presumably, well-insulated. After all, at a latitude of 71˚N, winters in Khatanga are dark and cold, with an average low of around –35˚C and no sun at all for many days in December and January. We were there in July and August, however, and the temperature was in the agreeable range of 5˚–15˚C, with sunshine twenty-four hours a day. Of course, there were a few mosquitoes hanging around, sullying the otherwise pleasant atmosphere. A few hundred mosquitoes, that is.
Per milliliter of air.
Our expedition team included Bernard, his wife Sylvie, and their twelve-year-old nephew Pitou; several Russians who worked for Bernard; a French filmmaker and her boyfriend; and a collection of academic scientists with a variety of interests in ice age animals. The most senior scientist in our team was Dan Fisher, a mammoth specialist and professor at the University of Michigan. Dan is a world expert in deducing everything about a mammoth—its sex and reproductive history, its lifestyle, and even how it died—by studying the growth patterns in its tusks. Dan also measures stable isotopes of elements, such as carbon and nitrogen, that are incorporated into the tusk as it grows. These isotopes contain a near-continuous record of changes in the mammoth’s diet and the environment in which it lived. We also had Adam Rountrey and David Fox, both of whom had trained under Dan’s supervision during earlier stages of their careers. And there were two of us interested in DNA: Ian Barnes, who at the time was a professor at Royal Holloway University of London but whom I knew from my time studying for my PhD at Oxford University, and me.
Dan, David, and Adam were keen to find tusks, and Ian and I were hoping for bones. Tusks are more useful for isotopic studies, but they contain very little DNA. Ian and I were also interested in the entire community of animals that had lived in the Taimyr during the ice ages, so we were not focused strictly on collecting mammoth bones.
For reasons that remain a mystery to me, and despite promises made to Bernard prior to our arrival in Khatanga, the helicopter was not available for a full week after we arrived. And so we waited. To pass the time while we camped out at Bernard’s, we explored Khatanga. We tried on various warm coats and mosquito-thwarting bits of gear. We wandered the streets, taunting local dogs and guessing what the purpose of the various machines might be. We practiced setting insect traps and identifying the things we caught. We drilled holes in a few bones from Bernard’s collection for the benefit of the film crew and future research projects. While we waited, Bernard arranged and was occupied by meeting after meeting with his team of Russian scientists and logistical experts. These meetings were colorful and exciting: giant maps were rolled out across tables that were too small to hold them, voices were raised, old scientific papers that outlined the geographical limits of past glaciations were consulted, vodka was consumed, and the excursion was planned.
Finally, the helicopter arrived, and it was time for us to get out into the field. We collected our food, fuel, and gear and headed from Bernard’s over to the airport. We maneuvered our way through security to the tarmac and came face-to-face with our next mode of transport: a well-loved Mi-8 helicopter. Two very large gas tanks already occupied about 25 percent of its interior. Working around the tanks, we packed in our camping gear, the cameras and lights for filming, two massive inflatable boats and 250 horsepower outboard motors, enough rice and freeze-dried anonymous foodstuff to feed twenty people for six weeks, a giant petroleum tank for cooking, and enough vodka to keep us happy for at least twenty-four hours. The Mi-8 was missing about a third of its windows, presumably to make smoking onboard more pleasant.
After loading was complete, we climbed aboard and settled in along the benches beneath the windows and on top of the gear and gas tanks. Last to board was Pasha, the cook’s dog. Pasha was a one-year-old Siberian husky and was communicating his apprehension about joining the expedition by attempting to fuse with the tarmac beneath the boarding stairs. Pasha and I were on the same page about which was a better fate: being swallowed by the tarmac or taking off in the Mi-8. When it became apparent that the tarmac would not absorb him, Pasha bolted. The cook and one of the pilots climbed out, smoked a few cigarettes, caught Pasha, manhandled him about halfway up the stairs, somehow let him escape, caught him again, subdued him sufficiently to get him all the way up the stairs and through the door, and finally we were set. To the sound of a few cheers and Pasha’s desperate howls, we lifted off the ground and headed into the tundra.
SOMATIC CELL NUCLEAR TRANSFER
If so many bones are already housed in collections across the globe, why go out into the field to find more? Why deal with broken helicopters, gold mines, twenty-four-hour daylight, and an infinite number of mosquitoes? The answer is simple: the best bones are those that come straight out of the frozen tundra. We want to find bones that have never thawed. These bones will contain the best-preserved cells, and those cells will contain the best-preserved DNA.
We are not the only team of scientists who spend our summers out in the Arctic searching for the remains of ice age animals or hanging out at placer mines, but I like to think that we are among the most realistic. We know, for example, that we are not looking for cells to clone. What scientists know about cloning animals from somatic cells—cells that are neither sperm nor eggs—suggests that cloning works only when they have a cell that contains an intact genome. No such cell has ever been recovered from remains of extinct species recovered from the frozen tundra.
Degradation of the DNA within cells begins immediately after death. Plant and animal cells contain enzymes whose job it is to break down DNA. These enzymes, called nucleases, are found in cells, tears, saliva, sweat, and even on the tips of our fingers. Nucleases are critical to us while we’re alive. They destroy invading pathogens before they can do any damage. They remove damaged DNA so that our cells can fix what’s broken. And, after our cells die, they break down the DNA in these dead cells so that our bodies can more efficiently get rid of them. This means that nucleases are evolved to remain active after a cell is no longer alive, which is bad news for cloning mammoths.
In the lab, we stop nucleases from degrading away the DNA we’re trying to isolate either by dropping a fresh sample in a solution of chemical inhibitors or by subjecting the sample to rapid freezing. The Arctic is a cold place, but not cold enough to freeze something—in particular something as large as a mammoth—quickly enough to protect its DNA from decay. In addition, all living organisms make nucleases, including the bacteria and fungi that colonize decaying bodies of dead animals. There is little chance, therefore, for any cells to retain completely intact genomes for very long after death. Without an intact genome, there will never be a cloned mammoth. That is to say, there will never be a cloned mammoth via somatic cell nuclear transfer.
Figure 8. Somatic cell nuclear transfer, or “cloning.” A tissue cell (top left) and unfertilized egg cell (bottom left) are harvested from different individuals. The nuclei are removed, and that from the tissue cell is transferred to the enucleated egg. An electric current is applied, and the egg begins to develop. The embryo is implanted into a surrogate mother and develops into an identical genetic copy of the tissue-cell donor.
Somatic cell nuclear transfer is a dull but appropriate name for the process that brought us, most famously, Dolly the sheep (figure 8). Dolly was cloned in 1996 by scientists at the Roslin Institute in Scotland. Scientists removed the nucleus, which is the part of the cell that contains the genome, from a mammary cell that they harvested from an adult ewe and inserted that nucleus into a prepared egg cell from a different adult ewe. The egg then developed, within the uterus of yet another adult ewe, into a perfectly healthy ewe. Importantly, the ewe that was cloned by nuclear transfer was genetically identical to the ewe that donated the mammary cell and nothing like the surrogate mother or the ewe that donated the egg.
To understand the intricacies of this process, we have to learn some basic facts about the cells that make up living organisms. Our bodies (and the bodies of other living things) are made up of three basic categories of cells: stem cells, germ cells, and somatic cells. Most cells are somatic cells, including skin cells, muscle cells, heart cells, etc. Somatic cells are diploid, which means they contain two copies of each chromosome—one from mom and one from dad. Somatic cells also have specialized roles: they might be brain cells, blood cells, or mammary cells like those that were used to create Dolly. Another category of cells is germ cells. Germ cells give rise to gametes, which are sperm and eggs. Sperm and eggs are haploid, which means they have only one copy of each chromosome. In normal sexual reproduction, the two haploid gametes fuse upon fertilization to create a diploid zygote, which then develops into an embryo.
In nuclear transfer, the fertilization and fusion step is skipped. Instead, the haploid genome of the egg (a germ cell) is removed in a process called enucleation. Then, a diploid nucleus from a somatic cell (in the case of Dolly, a mammary cell) is inserted in its place.
In normal mammalian sexual reproduction, the zygote that is formed at fertilization contains cells that are not at all specialized. These unspecialized cells fall into the third category of cells: stem cells. The stem cells found within early zygotes are called totipotent stem cells because they can become any type of cell and are therefore capable of creating an entire living thing. As development proceeds, these cells multiply and begin to differentiate, or to take on more specialized roles in the body. Very early in development, totipotent stem cells lose the ability to become every type of cell but are still very unspecialized. These cells are now called pluripotent stem cells. Pluripotent stem cells of mammals, for example, can become any type of cell in the body but cannot become placental cells.
Pluripotent stem cells have been of particular scientific interest from a therapeutic perspective. When stem cells divide, they can either make new stem cells or they can become specialized somatic cells. This means that they have the potential to replace cells that have become damaged or diseased. Stem cells are not only found in a developing embryo but are also found in tissues throughout the adult body. Adult stem cells tend to be more specialized than embryonic stem cells but are nonetheless critically important in tissue repair and replenishment. Many medical applications of stem cell therapy make use of adult stem cells. Hematopoietic stem cells, for example, can differentiate into various types of blood cells and are used to treat different forms of blood diseases, including leukemia.
Now back to cloning by nuclear transfer. Somatic cells, unlike stem cells, are highly specialized. Somatic cells cannot differentiate into different types of cells. They are the end of the line, as far as differentiation goes. Somatic cells have a particular job to do, and their cellular machinery is fixed in a way that makes them good at that job. In a somatic cell from a sheep mammary gland, only those proteins required to be a mammary cell are expressed, so only the genes that make those proteins are turned on.
In order for that somatic cell to become an entire living organism, it has to “forget” all of this specialization and de-differentiate. It has to turn back into an embryonic stem cell.
While Dolly is perhaps the most famous animal born via somatic cell nuclear transfer, she was not the first clone to be produced in this way. In the 1950s and ’60s, John Gurdon of Oxford University showed that frog eggs would develop into frogs even after their nuclei were removed and replaced with nuclei from somatic cells. Although the mechanism was not well understood at the time, Gurdon’s key observation was that the egg somehow triggered de-differentiation of the somatic cells—they forgot what type of cell they were. In 2012, Gurdon shared the Nobel Prize for these discoveries with Shinya Yamanaka of Kyoto University, who later discovered that the same pluripotency (de-differentiation of somatic cells) that was induced by the egg could be induced in vitro, that is, in tissue culture in a lab rather than in an egg, by adding a suite of transcription factors, which are proteins that bind to specific DNA sequences and control what genes are turned on and when. Such cells are known as induced pluripotent stem cells, or iPSCs.
Nuclear transfer has been used to clone sheep, cows, goats, deer, cats, dogs, frogs, ferrets, horses, rabbits, pigs, and many others. Cloning animals with specific sought-after traits is also gaining popularity. Commercial services to clone pets and to provide cloned offspring of champion horses are advertised widely on the Internet. The results of such selective cloning have begun to manifest: in late 2013, Show Me, a six-year-old clone of a polo-playing mare named Sage, won the Argentinian Triple Crown, perhaps ushering in a new era of animal breeding for show and sport.
Cloning by nuclear transfer is not particularly efficient, however. Dolly was the only one of 277 embryos created by the Roslin Institute that survived to be born. The first cloned horse to be born, a female named Promotea, was the only one of 841 embryos to fully develop. Snuppy, an Afghan hound cloned by the Korean scientist Hwang Woo-Suk, was one of two puppies born after 1,095 embryos were implanted into 123 different surrogate mothers, and the only puppy to survive for more than a few weeks. In each of these cases, scientists had access to a potentially limitless supply of somatic cells, all harvested from living animals.
There are no living mammoths.
SEARCHING FOR A MIRACLE
In the last several decades, sites rich in extremely well preserved frozen bones have been discovered across Siberia, Alaska, and Canada’s Yukon Territory. This area, collectively known as Beringia, was an important conduit for movement between Asia and North America during the Pleistocene. Based on the number and variety of bones collected from across Beringia, the area was teeming with megafauna—animals that weigh more than forty-five kilograms—throughout the Pleistocene. The remains of the Beringian megafauna are exposed when the permafrost in which they are buried is disturbed. We disturb the permafrost by building towns, building roads to connect the towns, and looking for gold. Ice age bones are also exposed through natural processes, such as the annual flooding of rivers and lakes after the spring snow melts (plate 10). High- and fast-flowing water rips around river bends, tearing into the frozen dirt along the river edges and washing out any bones or other megafaunal remains that had been frozen within the dirt.
Up in the Taimyr, Bernard selected a site for our base camp that he felt was a prime location for bone hunting, based on the hours he spent consulting maps and conversing with locals. We pitched our tents near the top of a relatively high, large hill within a landscape that was mostly water separated by patches of low-lying, treeless tundra (plates 11–13). Our plan was to walk the perimeters of the many lakes and connecting waterways, keeping our eyes peeled for bones or tusks.
I have spent many summers of my life searching for ice age bones in Beringia. Mostly it’s a lot of the same: wandering along rivers and lakes staring into the shallow water, or hanging out at active mining sites and waiting for the hoses to be turned off so that we can scan the freshly thawing surface for ice-age treasures. And nearly every day that I have spent in the field has been wildly productive.
Our first day in the Taimyr was unproductive. We set up our own tents, the cook’s tent, and our “rest” tent, which was really just a frame set inside a giant mosquito net that gave us enough space to crowd around a table away from the bloodthirsty onslaught. We inflated the boats and got them ready to go. We set traps to catch fish. We scoped out the edges of the lakes that were closest to us. We ate rice and fish, and celebrated our arrival in the field with a toast. And we found zero bones.
The second day was also not productive. We took the boats out and walked along the edges of lakes that were slightly farther away. We donned chest waders and ventured deeper into the freezing water. We found no bones. We returned to camp, and ate a dinner of rice and fish.
The third day was also unproductive. We split into smaller groups to scout out different nearby lakes, but nobody had any luck. That night, we sat in silence in our mosquito-free enclosure, eating our rice and fish. I’d never been on an expedition for three days and not found a single bone. I think the same was probably true for all of us. The glamour of being on an arctic expedition had pretty much worn off after the first seven thousand mosquito bites, and we’d already finished the vodka. To say the mood was bleak would be an understatement. Here we were, facing at least another several weeks on the tundra, with no idea why there were no bones to be found and no sense of what to do about it.
And then two things happened. First, we heard a rustle outside the enclosure and looked up to see two men who were not part of our expedition team standing there, quietly, with shotguns. Then, the French couple opened their cooler.
RENEWED HOPE AND THE BEASTS OF THE UNDERWORLD
More mummies have been recovered from permafrost deposits in Siberia than from permafrost deposits in North America. This may be because mammoth populations were larger in Siberia or because some aspect of the climate makes preservation of mummified bodies more likely in Siberia than in North America. Whatever the reason, the discovery of a mammoth mummy always causes a stir. For many of the indigenous people of the Siberian tundra, that stir is deeply personal. Some cultures have mythologies that refer to mammoths as beasts of the underworld and caution that touching them will bring bad luck—even death—to the unfortunate discoverer. More widely, though, the stir is one of excited anticipation. A mummified carcass is a special thing—and one for which scientists may be willing to pay a high price.
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 used 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.
It was this experiment that motivated Iritani and his team to try to clone cells from the Yukagir mammoth’s leg. Although Iritani’s team was unsuccessful (none of the mammoth cells developed to a stage where it was possible to try to make cell lines), he remains undeterred. His team had, after all, managed to isolate a nucleus from a mammoth cell, which was a remarkable feat in itself.
In August 2011, a mammoth thigh bone was found in the Sakha Republic that was so well preserved that it still contained greasy bone marrow. Certain that this was the ticket to cloned mammoths, Iritani used this find as a springboard to reinvigorate his mammoth-cloning plans. That December, Iritani announced that he would clone a mammoth by 2016. His timeline required that (1) they find a perfectly preserved mammoth during the following field season; and (2) they would be able to establish cell lines from that mammoth immediately. Given that elephants have a 600-day gestation period, his plan left no room for error.
Iritani’s announcement was embraced by the global media, which delighted in the opportunity to publish yet another round of mammoth-cloning-is-inevitable stories. The most intriguing response, however, came from South Korea, where one more contestant in the race to clone a mammoth was about to emerge.
In March 2012, Hwang Woo-Suk at the Sooam Biotech Research Foundation announced with great fanfare that Sooam had established a new collaboration with the North-Eastern Federal University in Sakha (with which the Mammoth Museum is affiliated, and with which Iritani had been working since 1997), and that he was going to clone a mammoth. The announcement went viral, complete with pictures of a smiling Hwang shaking hands with Vasily Vasiliev, vice-rector of North-Eastern Federal University, over official-looking documents. Almost immediately, the Moscow News published a clarification of the report. Without identifying its source, the Moscow News stated in strong and clear language that while the Russian Academy of Sciences certainly was planning to clone a mammoth, it would do so in collaboration with Iritani and the team from Kinki University and not with Hwang.
It is not surprising that reaction to Hwang’s involvement in the high-profile cloning project would bring about mixed emotions. I mentioned Hwang earlier in this chapter, with a brief reference to his work to produce the first cloned dog, Snuppy. Hwang is, however, better known for his work in human cloning. In the early 2000s, Hwang was leading a research group at Seoul National University that was at the absolute cutting edge in human stem cell research. His group published two major breakthrough papers in 2004 and 2005. The first claimed that they had cloned the first human embryos, and the second indicated that they had made stem cells that were genetically matched to specific people; these were enormous advances for biomedical research. In Korea, Hwang was praised widely as a national hero. And then the walls came crumbling down. In 2006, Hwang retracted both papers after it was revealed that the data had been faked. He lost his job at the university and was stripped of his license to conduct stem cell research. He was also charged with fraud, embezzlement, and bioethics infractions and was eventually found guilty of the two latter counts.
Hwang’s trial lasted for three years from 2006 to 2009. During this time, he joined the Sooam Biotech Research Foundation and continued his research, which was now focusing on cloning animals. The first official mention of Sooam’s plans to clone a mammoth came in 2012, with the announcement of collaboration with the North-Eastern Federal University. Hwang’s interests were already well established by that time, however. During his trial in 2006, Hwang explained why so much of the standard documentation of research expenses was missing from his files: he needed to pay the Russian Mafia for access to the best mammoth carcasses.
In the autumn of 2012, on the heels of their big announcement, Hwang Woo-Suk and his student, Hwang Insung, joined Semyon Gregoriev of the North Eastern Federal University on a three-week expedition up the Yana River to find a mammoth to clone. The trip was being filmed for National Geographic by a London-based documentary maker who intended to tell the story of Sooam’s project from start to, well, start. Although the expedition did not succeed in finding a mammoth mummy, reports emerged as soon as they returned from the field that a remarkably well-preserved piece of skin had been found buried in the frozen ground. Most importantly, the skin was said to contain cells with intact nuclei.
A few weeks prior to the trip, the filmmaker contacted me about joining the expedition as the genetics expert. Unfortunately, I had to stay behind (to give birth to my second son), but recommended my friend and colleague, Love Dalén, who runs an ancient DNA lab at the Swedish Museum of Natural History. Love tells a slightly less fantastic rendition of the story than the documentary portrays. In Love’s version, the team already knew where to look for a mammoth before the expedition began. Yakutian mammoth hunters had spent the early part of the season looking for tusks along the river. In doing so, they blasted a series of long tunnels into the permafrost along the riverbanks using high-pressure water. At the end of one such tunnel, someone had spotted a perfectly preserved baby mammoth. The mammoth—sans tusks, of course, as these would already have been removed by the first people to encounter the freshly exposed mummy—was still in place, and the plan for the show was to go back and get it. Unfortunately, by the time the expedition team arrived and filming began, late season rains and flooding had caused the tunnel to collapse, leaving the expedition/documentary team desperately searching those tunnels that remained for anything that would suffice for their show. The skin in question was found by Hwang Insung after he maneuvered into one such tunnel despite warnings from the expedition’s safety officer that it was dangerous to do so. Hwang found the piece of skin deep within the tunnel, just before getting word from the outside that the tunnel was about to collapse. After a few moments of desperate panic, those who had dared enter the tunnel emerged, narrowly escaping being crushed by several thousands of kilograms of frozen dirt.
Did the bit of mammoth skin that they found have cells that contained intact nuclei? Perhaps. Finding what appears to be cellular structure is not uncommon in permafrost-preserved remains. Will the genome within those cells be sufficiently intact to be cloned? Doubtful. Love was able to take a subsample of the specimen to Stockholm where he extracted and amplified DNA. He confirmed that the skin was, in fact, from a mammoth. But the longest fragments of DNA that Love could amplify were around 800 nucleotides long. That is a remarkably long fragment for ancient DNA (the average length of fragments from permafrost-preserved specimens is closer to seventy nucleotides long), and it certainly indicates that the specimen is well preserved. Still, 800 nucleotides is a far cry from the length of an intact chromosome.
In the summer of 2013, a new partial mammoth carcass was discovered frozen in a lake on Malolyakovsky Island, part of the New Siberian Islands. The find was absolutely stunning. The part of the mammoth that had been exposed was beginning to rot, but other bits of flesh were so well preserved that they were described as looking like fresh meat. Most intriguingly, a deep red substance suspiciously reminiscent of blood was found in the permafrost beneath the carcass. While most experts (myself included) are highly skeptical that the substance actually is blood—there is no animal with blood capable of staying unfrozen in the conditions in which this sample was recovered—research has been inconclusive so far with regard to what it actually is. The specimen has been kept frozen and is currently being studied in Yakutsk by scientists from around the world.
Is this latest mammoth the “best preserved mammoth in the history of paleontology,” as Semyon Gregoriev, who led the expedition to recover its remains, is quoted as having said? Dan Fisher was one of the first to examine the specimen, and he confirms that parts of it are indeed impeccably well preserved. As to whether it is sufficiently well preserved to contain intact nuclei, we will have to wait and see. I remain skeptical.
AND SO THE SEARCH CONTINUES
It so happened that the two men who appeared suddenly outside of our enclosure on the third day of our ill-fated Taimyr expedition were related to the Jarkovs—the family that found and alerted Bernard of the Jarkov mammoth in 1997. They were Dolgans, a group of people who are indigenous to that part of the Taimyr. While the rest of us were trying to pretend that the sudden appearance of strangers with guns had not nearly caused us to have simultaneous heart attacks, Bernard was inviting them into the enclosure and exchanging hearty handshakes and bises. Bernard, it appears, knows everyone in Siberia.
Dolgans are nomadic reindeer herders. During the summer months, they move around the tundra, allowing their large herds to graze. They settle in one spot for a few weeks, until the reindeer have eaten everything in sight, and then pack up and move to the next place. In doing so, they have a chance to scope out pretty much the entire region. If bones, tusks, or mummified mammoths had been exposed when the ground thawed that spring, the Dolgans would know about it. The two men who joined us had seen our helicopter fly in a few days earlier and were curious to know what was going on. So, while the rest of their families were packing up to move on to their next location, the pair set out in search of us.
As the initial shock of the men’s surprise appearance wore off, the heaviness that had settled in among the members of our expedition team began to lift and be replaced by the familiar, excited anticipation of what was to come. We gave them all the fish and rice they could eat and apologized for the lack of vodka. When the French couple opened the cooler and pulled out two giant cheeses—a gouda the size of a human head and what must have been three kilograms of brie—the entire crew erupted into laughter. Of course a French family working alone in Siberia would have a cooler filled with cheese. Even Pasha, who had managed to inch his face into the enclosure in a desperate attempt to keep the mosquitoes out of his nose, sniffed and flopped his tail onto the tundra. The whole scene was completely absurd, and we were only on day three.
We invited the Dolgan men to stay in our camp for the night and, the next morning, took them back to their families in our outboard-powered inflatable boats. The entertained us for a while; we chatted about the weather, shared some French cheeses, and ate some of their prepared dried fish. We asked whether any of the Dolgans knew of sites that were actively producing bones. They had a few ideas but no strong leads. Then they finished packing up, hooked up their houses and gear to the reindeer, and set off for their next stop on the tundra.
During the rest of the summer, we found only a few scraps of mammoth bone, as well as intact but poorly preserved bones from horses, steppe bison, and woolly rhinos. We later learned that the area we were searching had been covered by ice for most of the Pleistocene, which explains our lack of success. Luckily, before we left Siberia, Ian and I were able to take samples from some extremely well preserved bones that had been collected during previous years’ expeditions and that were stored in Bernard’s collection in Khatanga, so the trip was not entirely wasted.
These bones did not contain cells with intact genomes. Fortunately, however, perfectly preserved genomes are not critical to de-extinction.
