Plate 11. A first look at base camp. Ian Barnes and I pose for a photograph as the helicopter is unloaded on the Taimyr Peninsula in the Russian Far North. Other members of the 2008 expedition team have already donned their mosquito-net hats. Photo credit: Beth Shapiro.
Plate 12. Setting up and settling in. As the mosquitoes swarm overhead, the 2008 Taimyr expedition team begins to stake out its tent sites. Our site is at the top of a hill surrounded by lakes, all of which we will search over the next weeks for the remains of mammoths and other ice age animals. Photo credit: Beth Shapiro.
Plate 13. Another shot from the first day of our 2008 Taimyr expedition: my tent, and several million mosquitoes. Photo credit: Beth Shapiro.
Plate 14. An ice cave beneath the city of Yakutsk, Sakha Republic, Russia. Caves such as this one are often used in Siberian cities to store food during the summer months. At the far end of this ice cave, scientists prepare to display the Yukagir mammoth to a delegation of international scientists attending a conference. Photo credit: Beth Shapiro.
Plate 15. Wild Spanish ibex escape manipulation by the scientists leading the bucardo-cloning project. Accustomed to climbing vertical rock faces and balancing on narrow ledges, the wild ibex easily balance atop a thin ledge within the captive breeding facility, well out of reach of the research team. Photo credit: Alberto Fernández-Arias.
Plate 16. Grazed and ungrazed land in Sergey Zimov’s “Pleistocene Park” in the spring, after snowmelt. Ten years earlier, the area was a continuous community of willow shrubs. Today, the grazed area (foreground) in early spring has small amounts of green grass and freshly churned soil. This is caused by herbivores returning to this site during winter to graze and, in the process, trampling the snow and exposing the soil to the cold winter air. Photo credit: Sergey Zimov.
CHAPTER 8
NOW CREATE A CLONE
I have, up until this point, been very clear that mammoths will not be resurrected by cloning. What I say next, therefore, is likely to be confusing. The next step in bringing a mammoth back to life is to create a clone.
In my defense, the cells that we would clone at this stage would be very different from the cells that the Japanese and South Korean teams hope to find and use in their cloning experiments. By the time we arrive at this stage of de-extinction, we might have spent years (even decades) in the lab, painstakingly engineering changes to the elephant genome within our cells. We would not be beginning our cloning experiments with miraculously well-preserved mammoth cells. Nonetheless, the next step in de-extinction would be to “clone” our cells, and thereby turn them into an entire elephant (with some mammoth genes).
Some de-extinction projects will, of course, be able to skip the genome-engineering steps and proceed directly to cloning. These projects may move ahead much more quickly than those that require genome engineering. Of course, that simply means that they will be first to arrive at the next hurdle. Consider the example of the bucardo.
NOT QUITE THE FIRST DE-EXTINCTION
In the summer of 2003, a young female bucardo, which is a subspecies of Spanish ibex (a type of wild goat), was born. Bucardos had been endemic to the Pyrenees, the mountain range that forms the border between Spain and France. When this baby bucardo was born, however, bucardos had been extinct for just over three and a half years.
The baby bucardo was a genetic clone of the last living bucardo, an elderly female named Celia. Unfortunately, the baby suffocated within minutes of birth. An autopsy revealed that she had been born with a malformed lung and had no chance of survival. Nonetheless, the birth of this baby bucardo is often held up as the first successful de-extinction. I disagree. To me, if she had no chance of survival, this is not de-extinction.
As de-extinction projects go, the bucardo project has a lot of promise. Bucardo cells were harvested from Celia ten months prior to her death and immediately frozen, and the DNA within those cells is in very good condition. Several closely related subspecies of Spanish ibex are still thriving, so finding appropriate egg donors or surrogate mothers should be straightforward. The bucardo has also not been extinct for very long, and its extinction was likely due to overhunting and not to the disappearance of its habitat. As long as we can control our guns, resurrected bucardos could be returned to their native habitat without the need for extensive environmental impact studies or political maneuvering.
When the team of Spanish and French scientists began the bucardo project in 1989, bucardos were not yet extinct. Cross-species cloning had also not yet been achieved in large mammals, and the challenges facing the project were immense.
In 2001, and in a separate effort to perform cross-species cloning, the biotechnology company Advanced Cell Technologies successfully cloned a gaur, which is an engendered species of cattle native to South and Southeast Asia, using a cow as a surrogate host. The cloned gaur lived only forty-eight hours before succumbing to dysentery, but its birth demonstrated that cross-species cloning was possible. Two years later, the same company successfully cloned a banteng, another endangered cattle species from Southeast Asia, again using a cow as a surrogate host. This banteng lived in the San Diego Zoo for seven years—less than half its lifespan in the wild—before dying of what appeared to be natural causes.
The bucardo project was similar to the gaur and banteng projects in that there was no need for genome sequencing or genome engineering for cloning to be possible, and in that surrogate hosts were available. There were, however, two important differences that distinguished the bucardo project from the others. First, assisted reproductive technologies were already established for cattle, but had not yet been developed for Spanish ibex. Second, by the time the team of scientists developed this technology, the bucardo had gone extinct.
Unfortunately, the bucardo cloning experiment did not succeed, and it is not entirely clear why. It is possible that the experiment failed because the scientists simply did not make enough embryos. Cloning by nuclear transfer is, after all, notoriously inefficient. The team transferred copies of Celia’s somatic cells into 782 eggs, but only 407 eggs developed into embryos. Of these, 208 embryos were transferred into potential surrogate hosts, but only seven pregnancies were established. Only one pregnancy lasted to term, and the baby bucardo that was born lived for less than ten minutes. If one were to count this baby bucardo as a successfully established clone, which I will do here only for the purpose of illustration, the success rate of bucardo cloning would be 0.1 percent.
Alberto Fernández-Arias, who is the director of the Aragón Hunting, Fishing, and Wildlife Service and who was brought in to the bucardo project in 1989 to develop assisted reproductive technologies in Spanish ibex, believes that I am being unfair in my characterization of this as a “failed” de-extinction. He points out that if his team had known that the bucardo would be born with a lung deformity, they could have been prepared to remove the malformed part of the lung immediately after birth. Such surgery has been performed successfully on human babies with similar birth defects and, indeed, may have been able to save the baby bucardo’s life. Of course, it is not possible to know either what caused the lung deformity or what might have happened—how the bucardo would have developed, how it might have fared in adulthood—if the bucardo had survived. The project continues, however, and we may soon have another opportunity to find out whether bucardos will once again roam the Pyrenees.
De-extinction by Nuclear Transfer
Once we have a cell that contains the genome of the animal we want to create, whether that cell has been grown from frozen tissues that were harvested prior to extinction or subjected to genome editing, the next step is to create an embryo from that cell. This involves using a living host as a surrogate. In many candidate species for de-extinction (I will describe some exceptions later in this chapter), this involves cloning by nuclear transfer. And, as one might anticipate, some candidate species for de-extinction will be considerably easier to clone than others will be. Cloning the bucardo, for example, should be much si
mpler than cloning edited elephant cells would be. For that reason, I will begin the exploration of this phase of de-extinction using the bucardo as an example. Once the basics have been covered, I will move on to the bigger challenges to be faced when cloning genetically engineered elephant cells. And, finally, I will introduce an obstacle to de-extinction that took me completely by surprise: it is not possible to clone birds.
The Making of a Bucardo
Nuclear transfer is a complicated process with potential disaster lurking in each step. Even what should be the simplest steps can harbor significant obstacles. With dogs, for example, it is nearly impossible to harvest mature eggs—that is, the eggs into which the somatic cell would be transferred—from female dogs. Unlike the eggs of other mammals, which mature in the ovary, dog eggs mature as they move from the ovary into the uterus. Because domestic dogs also tend to have unpredictable ovulation cycles, knowing when to harvest mature eggs requires both careful monitoring of the dog’s hormones and a bit of luck.
The most challenging step in nuclear transfer is, however, reprogramming. During reprogramming, the cell forgets how to be a somatic cell and becomes, essentially, an embryonic stem cell. Only cells that have reset completely can later differentiate into all of the various cell types that make up an organism. This step is, however, particularly inefficient. Incomplete reprogramming is thought to explain why so few embryos develop after nuclear transfer and the high frequency with which developmental defects are observed among those embryos that do develop.
Reprogramming is not the only step that can fail. Even if cells are reprogrammed correctly and develop into viable embryos without developmental defects, the egg may fail to implant in the uterus of the surrogate mom, or the pregnancy may fail after implantation. This could be due to a poor understanding of the reproductive cycle or to some kind of incompatibility between the developing embryo and the surrogate mother. Such incompatibilities are likely to be more common among cross-species clones (including de-extinction experiments, where all or part of the genome is from a different species than the surrogate mother) compared with same-species clones. Also, there is little doubt that experimental manipulation is stressful for the surrogates and that this stress may contribute to the elevated rate of failed pregnancies in cloning experiments.
Anxious Ibexes and a Hybrid Solution
Stress was certainly a limiting factor in the bucardo cloning experiments.
In preparation for working with bucardo cells, the scientific team leading this work first attempted a cross-species cloning experiment with a different and relatively common subspecies of Spanish ibex. Once these technologies had been developed and fully tested, the team would proceed with the bucardo.
The plan required Spanish ibex embryos. To create these embryos, the scientists first had to capture Spanish ibex from the mountains. Then, they needed to rear the captured ibex in captivity to observe their reproductive behavior and develop a way to make the females ovulate. After mating had been observed, the scientists would harvest fertilized eggs, implant the developing embryos into domestic goats, and hope for the best.
Harvesting Spanish ibex eggs turned out to be much more difficult than the team anticipated. Accustomed to climbing the steep faces of rocky slopes, the Spanish ibex escaped manipulation by taking refuge along high ledges in the walls of the animal facility (plate 15). When the team finally harvested their eggs, the eggs were all unfertilized. The animals, it seemed, were too stressed by the captive breeding environment to mate successfully.
The team was able to develop less stressful ways of manipulating the ibex and, eventually, they recovered fertilized eggs from captive Spanish ibex. Any excitement derived from this success was short-lived, however, as another serious setback to the experiment was revealed: none of the embryos continued to develop after implantation in domestic goats. It seemed that the domestic goat uterus was incompatible with the Spanish ibex embryo. This was bad news for bucardo cloning.
Believing that genetics might be the solution, the team decided that a different surrogate mother—one that was genetically more similar to the developing embryo—might be just what they needed. The most genetically similar surrogate would be a subspecies of Spanish ibex. They knew, however, that Spanish ibex were difficult to manipulate and did not fare well in captivity. Not wishing to spend every day coaxing ibex down from the walls, they decided on a compromise: they would create hybrids. Female domestic goats crossed with male Spanish ibex would produce kids with 50 percent Spanish ibex DNA and, most crucially, that would probably keep their feet on the ground. When these hybrid females reached adulthood, they would become the surrogate mothers for the Spanish ibex embryos.
Around a year later, the team transferred Spanish ibex embryos into hybrid goat-ibex females and, again, hoped for the best. Excitingly, half of the embryos established successful pregnancies and developed into healthy Spanish ibex.
I should point out that this success rate—50 percent survival of implanted embryos—is high because this particular experiment did not involve nuclear transfer. This experiment began with healthy embryos taken from living ibex, not with somatic cells that needed to be reprogrammed. As I noted before, this reprogramming step—which is the first step in bucardo de-extinction—has an extremely low success rate.
Unanticipated Barriers to De-extinction
In developing assisted reproduction technology for Spanish ibex, the bucardo-cloning team learned that bucardo embryos, should they get that far in the bucardo-cloning experiment, might develop within surrogate mothers that were hybrids of domestic goats and Spanish ibex, but they were unlikely to develop within purebred domestic goats. The team had discovered a barrier of some sort to cross-species cloning that had arisen during the evolutionary divergence between these two lineages.
Importantly for de-extinction, the probability that barriers such as this may arise increases with evolutionary distance. Extinct species with no close evolutionary relatives might not have any suitable living potential maternal host. The ibex experiment revealed, however, that barriers can also exist between closely related species. Genome editing could even cause barriers—for example, by disrupting important interactions between the embryo and the maternal host. In this way, even de-extinction projects that involve only minimally edited genomes may be frustrated by unanticipated incompatibilities between the developing embryo and the surrogate host.
Some incompatibilities may manifest even before the implantation phase. If, for example, the egg cell into which the nucleus of the somatic cell is injected is incompatible with the somatic cell, then none of these eggs will develop into embryos even if the somatic cell is completely and correctly reprogrammed. Such a problem may arise, for example, if the nuclear genome from the somatic cell is incompatible with the mitochondrial genome in the egg cell.
Mitochondria are organelles that live within the cytoplasm of cells and are not part of the nuclear genome. All of the mitochondria that an organism will have in all of its cells are descended from the mitochondria in the egg cell. Mitochondria have their own genome, and this genome codes for some of the proteins that are necessary for cellular respiration—that is, the process by which cells use oxygen and sugars to make energy. Other proteins necessary for cellular respiration are made by genes in the nuclear genome. Incompatibility between the mitochondrial and nuclear genomes can lead to incompatibilities between these genes. If these genes can’t work together to make the cell respire, this can lead to metabolic disease, neurologic disease, and even death. Thus far, all cross-species cloning has involved the transfer of only nuclear DNA—not mitochondrial DNA.
Researchers in David Rand’s lab at Brown University demonstrated how nuclear-mitochondrial mismatch can produce unusual phenotypes in otherwise genetically normal cross-species hybrids. Rand’s lab created fruit flies with nuclear DNA from Drosophila melanogaster and mitochondrial DNA from Drosophila simulans, two fly species that diverged from each other around 5.4 million years ago.
The resulting mismatched-genome flies had whiskery bristles on their backs, were half the length of normal flies, were developmentally delayed, reproduced poorly, and, as might be expected if energy production is off, got tired more quickly than did flies with matched genomes.
Mismatches between the mitochondrial and nuclear genomes might be a problem for de-extinction, but not one without an obvious solution. If the mitochondria don’t match, why not replace them with mitochondria that do match the nuclear genome? Or, why not edit the mitochondrial genome to replace the problematic sites? This could presumably be achieved using the same genome-editing approaches as would be used to alter the sequence of the nuclear genome. Neither of these approaches is simple, and neither is feasible today. Both are, however, theoretically possible.
MAMMOTH PROBLEMS
Now that I’ve introduced some of the challenges to be faced during the cloning and prenatal development stage of de-extinction, let’s return to the mammoth as a specific example. As I discussed in the preceding chapter, we have the technology today to edit the elephant genome so that it contains the mammoth version of least some genes. Assuming that genome engineering takes place in a cell that either is a stem cell or can be reprogrammed to become a stem cell, we are ready to move on to the next step: creating a living animal that contains the edited genome and, hopefully, expresses the traits that we aim to resurrect.
To complete this step, the cell needs to develop into an embryo, and because we cannot grow an elephant in the lab, that embryo needs to be transferred into a surrogate host. Once inside the surrogate, the embryo needs to implant in the uterine wall and establish a pregnancy. The pregnancy then needs to proceed without problems and culminate in the birth of a healthy baby animal whose genome contains several carefully selected and painstakingly engineered mammoth genes.
How to Clone a Mammoth Page 16