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 t
able 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.
How to Clone a Mammoth Page 9