How to Clone a Mammoth

by Shapiro, Beth

  The simplest way to transform the edited cell into an embryo is to use an egg. We know that eggs contain proteins that activate cells—that is, they reset cells that have already differentiated and turn them into embryonic stem cells. The most appropriate egg to activate our edited elephant cell is, unsurprisingly, an elephant egg. Elephant eggs are not particularly easy to come by. When an Asian elephant ovulates, she releases only one egg at a time. Once released, the egg travels through her reproductive system to the uterus, which is, predictably, elephant sized. An elephant that is not pregnant will ovulate once every two to three months. Given the poor efficiency of nuclear transfer, it’s reasonable to assume that collecting a single egg every two months—assuming we can find that egg within the elephant’s reproductive tract—will not provide enough eggs. We’ll need hundreds, even thousands of elephant eggs for this to work. Frankly, that seems unfair. Elephants are struggling to make enough elephants to sustain healthy populations; the last thing they need is for us to be snooping around their ovaries stealing their precious few mature eggs. In fact, if harvesting mature eggs from adult elephants were the only way to get elephant eggs, my opinion would be that mammoth de-extinction research should stop immediately.

  Fortunately, there may be another way. In 1998, researchers at Purdue University and the Advanced Fertility Institute at Methodist Hospital of Indianapolis created mice that could grow elephant eggs. Dr. John Crister, who led the study, wanted to develop a way to increase the reproductive rate of endangered species, and he hoped that coaxing laboratory mice to grow their eggs would be a good start. He and his team transplanted ovarian tissue—the tissue in which immature eggs are found—that Crister collected from three South African elephant carcasses into several laboratory mice. A few of these mice developed egg-producing follicles and, ten weeks later, one of these follicles produced a slightly misshapen elephant egg. Crister and his team did not attempt to fertilize the egg with elephant sperm, so it’s not possible to say whether it would have developed into a viable embryo. It is, however, an optimistic start.

  Hopefully, scientists will invent an efficient means to collect a large number of elephant eggs without jeopardizing any elephants. We could then collect a ton (perhaps literally) of elephant eggs, remove their nuclei, and insert nuclei that contain our edited genomes. Then, we would sit back and allow the egg to perform its reprogramming magic. If this goes smoothly and we end up with viable, developing elephant embryos (with slightly modified genomes), we can then transfer these embryos into the uteri of adult female elephants, where they can develop into baby elephants (with slightly modified genomes).

  The entrance to an elephant’s uterus is blocked by a membrane called a hymen. In elephants, the hymen stays in place throughout pregnancy, ruptures during birth, and then grows back in preparation for the next pregnancy. To establish a healthy pregnancy in a surrogate elephant mother, the embryo and whatever tool is used to deliver it into the uterus must pass through the only opening in the hymen—a four-millimeter hole designed to allow only sperm to penetrate—without destroying the membrane and thereby compromising the pregnancy.

  Let us assume this is possible. Let’s also assume that the pregnancy takes hold, and the embryo begins to develop. The next step is to wait patiently while the pregnancy proceeds. A typical gestation period for an Asian elephant is somewhere in the realm of eighteen to twenty-two months. Hopefully, no compatibility issues will develop between the embryo and the surrogate mother during this time. Hopefully, the surrogate mother’s genetic makeup won’t influence the expression of the genes we changed. Hopefully, her diet, hormones, and stress level won’t alter the developmental environment in a way that influences the expression of the genes that we changed. Hopefully, the birth goes well for both the surrogate mother and the neonate.

  Size Matters

  In designing cross-species cloning experiments for the purpose of de-extinction, it is important to consider physical differences between the two species involved. Mammoths that lived during the Late Pleistocene varied considerably in size. The largest of these mammoths were about the same size as big African elephants and the smallest were similar in size or even smaller than average-sized Asian elephants. It is not known whether this size variation was genetically determined or simply reflected differences in the amount and quality of available food. Regardless, this variation might be important in choosing surrogate hosts. Interestingly, the two baby mammoth mummies that have been found were both around ninety centimeters tall, which is approximately the same size as a newborn Asian elephant, suggesting that the most closely related elephant species to a mammoth might make a reasonable surrogate.

  Physical differences in size can cause problems in gestation and birth, however. Imagine, for example, if sperm from a Great Dane was used to impregnate a Chihuahua. The embryos would begin to develop and fill whatever space was available, but development would stall as they ran out of room to grow. Ultimately, the embryos may die, the mother may die, or both may die. If a natural birth were attempted, the mother would almost certainly suffer terribly. Returning to de-extinction, what might happen if a very large auroch were to develop within a much smaller, domestic cow? Or if a dugong tried to gestate a Steller’s sea cow? Size differences between species, even between closely related species, should definitely be considered when proposing surrogate hosts.

  A possible solution might be to make miniature versions of some extinct species. We could identify which genes or suite of genes are most critical to determining body size and tweak these using genome editing. A useful clue about which genes to target could come from genetic analysis of the population of mammoths that lived on California’s Channel Islands. These so-called pygmy mammoths grew to only around two meters tall at the shoulder, compared with four meters or more for mainland mammoths, and probably weighed just under 800 kilograms, compared with 9,000 kilograms or more for mammoths on the mainland. There is one problem with this idea. Tiny mammoths may be easier to gestate, but they might not be sufficiently large to replicate the ecological interactions between normal-sized mammoths and the ecosystems in which these normal-sized mammoths lived. Resurrecting pygmy mammoths therefore might not achieve the environmental goals of mammoth de-extinction.

  Another potential solution is to give up entirely on surrogates and instead use artificial wombs. I’m imagining something similar to the artificial wombs that Aldous Huxley envisaged to grow children in his book Brave New World. Or, even better, the giant nutrient-filled flasks in which human clones were grown on the planet Kamino to fight for the good side in the movie Star Wars: Episode II. In the artificial womb scenario, embryos would develop to term in a completely artificial environment—an idea known as ectogenesis. Modern medicine is a long way from functional artificial wombs and successful ectogenesis, but there is little doubt that innovations in this realm would have considerable impact on neonatal and perinatal care. Plus, by using artificial wombs, any animal suffering caused by surrogacy would be avoided entirely. The use of artificial wombs assumes, however, that developing within a real uterus is not critical to normal mammalian development. This is something that science does not yet know.

  CLONING IS FOR THE BIRDS (NOT)

  Although my focus until now has been on mammoth de-extinction, the present discussion provides an ideal opportunity to shift to the other de-extinction project with which I am involved—resurrecting the passenger pigeon. I hinted earlier that some species would not be cloned using nuclear transfer. The passenger pigeon is one of those species.

  Because birds develop on the outside, rather than within the bodies of their surrogate moms, birds would seem to be a good choice for cloning by nuclear transfer. Yet, none of the species listed as having been cloned using this approach were birds. Why is that?

  The simple answer is that birds cannot be cloned in this way.

  A bird begins its long journey to becoming a bird as a yolk. The yolk is a single unfertilized cell—the oocyte—that li
ves inside the bird’s ovary. The first step in bird development is to release the yolk into the oviduct. As it begins its journey down this very long, very spirally tube, it meets sperm and is fertilized. Then, for the next twenty-four hours or so, the fertilized egg travels slowly through the oviduct, tumbling around dramatic twists and through spiraled coils. As it bobs along its path, layers of albumin and structural fibers gradually cover the egg. This is the stuff we know as egg white. As it is moving, the fertilized cell starts to divide. The egg tumbles through the oviduct, twisting the structural fibers around the yolk, anchoring it firmly within the egg white. Toward the end of the oviduct, and just before the egg is laid, the hard shell is deposited as the final layer around the developing embryo. By the time it completes the journey from inside its mother’s ovary to the outside world, the embryo comprises around 20,000 cells. These will have begun to differentiate into different cell types.

  At what point in this process would it be possible to perform nuclear transfer? In a mammal, the egg whose nucleus is removed and then replaced is collected from the female reproductive tract after it has matured but before it is fertilized. At precisely this stage, the egg is primed to reprogram the nucleus of the somatic cell. It turns out to be extremely challenging to collect bird eggs that are at this stage of development. The reproductive tract in birds is long and sinuous, and the yolk is tricky to recover prior to fertilization. If we wait until the egg has been laid, the cells in the embryo will have already started to differentiate, and the embryo—which is held in position within the egg by many layers of twisted fibers—will be too large to remove. Even if the embryo could be removed and replaced without destroying the egg, the replacement embryo would have to be at the same developmental stage as the egg’s natural embryo. Growing embryos to such a late stage in the lab is also proving to be extremely challenging. For the moment, therefore, it seems that cloning birds may never be possible.

  Fortunately, there is another way. When the bird egg is laid, the embryo is still in an early developmental stage. The primordial germ cells—those cells that will later develop into either the sperm cells or the egg cells of the developing embryo—have formed but have not yet found their way to the sex organs, as the sex organs do not yet exist. Around twenty-four hours after the egg is laid, the primordial germ cells migrate through the developing embryo’s bloodstream to the sex organs (which are now starting to develop), where they settle in until the time comes when they mature into sperm or eggs.

  Primordial germ cells are the key to genetically manipulating birds. Primordial germ cells can be grown in a dish in the lab, which makes their genomes accessible for editing. Primordial germ cells are also tiny, which means they can be injected into the egg during that second twenty-four-hour window of development during which the egg is on the outside and the primordial germ cells are making their way to the bird’s developing sex organs. In this way, the injected edited primordial germ cells will travel with the embryo’s primordial germ cells to the sex organs. When these cells mature, the edited cells will participate in making the next generation of birds.

  When the chick hatches from the egg into which the genetically modified primordial germ cells were injected, that chick itself will not be genetically altered. Instead, the genetically altered cells will be hiding out in its sex organs. The first time the genetically altered genes will be expressed will be when that chick grows up and has its own baby chicks.

  Let’s walk through how this process would work for passenger pigeon de-extinction. Band-tailed pigeons are the closest living relative of passenger pigeons. The intention of the passenger pigeon de-extinction project, although these experiments have not yet begun, is to create band-tailed pigeons that look and act like passenger pigeons. To achieve this, we will isolate primordial germ cells from band-tailed pigeons and grow these in the lab. We will then edit the genomes within the primordial germ cells using the genome-engineering technologies described several chapters ago, replacing band-tailed pigeon genes with the passenger pigeon version of these genes as appropriate. We will then inject these edited band-tailed pigeon primordial germ cells into developing band-tailed pigeon eggs at precisely the right time during development. The chicks that are born when these eggs hatch will be genetically pure band-tailed pigeons, except that some of their germ cells (sperm or eggs) will contain passenger pigeon DNA. The offspring created by these edited germ cells will contain passenger pigeon DNA throughout their bodies.

  Cloning by Germ Cell Transfer

  Cloning by transferring germ cells into a developing embryo has one important advantage when compared with cloning by nuclear transfer. Edited primordial germ cells do not need to be reprogrammed. This is huge. So why has all the focus been on cloning a mammoth, when cloning passenger pigeons or dodos would apparently be so much simpler?

  It is not entirely clear why cloning birds has received far less attention than cloning mammals has. Primordial germ cell transfer works remarkably well as a means to genetically modify birds. The technology has been developed mainly with the chicken industry in mind, but it has been used both for conservation purposes and in pure scientific research. There is no obvious reason to suspect it would not work well for the purposes of de-extinction.

  Some of the applications of primordial germ cell transfer are, admittedly, unusual. The Roslin Institute, the facility that was responsible for cloning Dolly, has used the technology to create chickens that glow a bright green color under ultraviolet light. To make their chickens glow, they insert a protein into their genomes called green fluorescent protein, or GFP, which is found naturally in the North American jellyfish Aequorea victoria. The scientific community uses GFP to track biological changes within an organism. For example, if tissues whose cells express GFP are grafted onto an organism whose cells do not express GFP, scientists can track what happens to the grafted cells by watching them under fluorescent light. Scientists interested in using glowing chickens for their research can go to the Roslin Institute’s Web site and order them. For now, there is no charge.

  In addition to making chickens glow, the technique of injecting primordial germ cells into developing embryos has been used to boost the population sizes of rare or endangered chicken breeds. Primordial germ cells can be harvested from the blood of developing embryos without killing the embryo. These cells can then be kept alive in the lab and introduced into the developing embryos of common breeds. When these birds reach sexual maturity, they can then be fertilized with sperm (which are much easier to collect than eggs) from the rare breed. When these sperm fertilize eggs that develop from the injected primordial germ cells, the result is a purebred rare-breed chicken that hatches from an egg laid by a common chicken.

  The most exciting application of primordial germ cell transfer from the perspective of bird de-extinction has been the successful transfer of primordial germ cells between species. Scientists at the Central Veterinary Research Laboratory in Dubai injected primordial germ cells from a chicken into duck eggs. When the ducks hatched from these eggs, they looked like perfect ducks. Remember, only the germ cells are different in the first generation. The scientists later harvested sperm from these ducks, and used those sperm to fertilize a hen. When the eggs laid by this hen hatched, perfect chickens were born. With a duck for a dad.

  Fascinatingly, ducks and chickens are not the only animals that have been coaxed to give birth to a different species using this approach. Recently, Professor Goto Yoshizaki of the Tokyo University of Marine Science and Technology transferred rainbow trout eggs and sperm into the reproductive tracts of adult Masu salmon. When these adults mated, some of their eggs hatched into rainbow trout. Rainbow trout and salmon are closely related species, which may explain the experiment’s success. However, there is hope that the technique can be extended to other fish species. Yoshizaki also produced tiger puffer fish using grass puffer fish and intends to use mackerel to produce bluefin tuna, which, if successful, would provide an inexpensive way to increase tuna
production without removing juveniles from the wild.

  Germ cell transfer is certainly an exciting technology, and one that may have a variety of uses in conservation biology. There are some drawbacks, however, to using germ cells for the purpose of de-extinction.

  First, primordial germ cells are haploid; they either become sperm or eggs. When a sperm with an edited genome fertilizes an egg that does not have an edited genome (or vice versa), the offspring’s diploid genome will have only one copy of the edited gene. The edits, therefore, may not be visible in the offspring’s phenotype. To produce offspring with two copies of the edited gene, genomes from both the sperm and the egg have to be edited.

  Second, the injected primordial germ cells are not the only primordial germ cells that make it to the sex organs. In the duck example above, the duck was the dad of the chicken, but his sperm were chimeric—some of his sperm were duck sperm and other sperm were chicken sperm. When his duck sperm fertilized a chicken egg, nothing happened. No hybrid “duckens” were born. But, when his chicken sperm—those of his sperm that were derived from the chicken primordial germ cells that were injected into his egg while he was a developing embryo—fertilized a chicken egg, a chicken was born. That chicken had a genome that was 100 percent chicken-like. But its father was, nonetheless, a duck.

  Third, in the experiments that have been done thus far, scientists observed that the efficiency with which the injected primordial germ cells go on to become the next generation is poor. Only a small fraction of the eggs and sperm that are eventually made by the embryos develop from the injected primordial germ cells.