RARE BEAUTY: The jaguar (Panthera onca) is one of the threatened wild species that researchers in Brazil may try to clone. Image: James and snowmanradio, Wikimedia Commons
In 2009 the Brazilian Agricultural Research Corp. (Embrapa) and the Brasilia Zoological Garden began scavenging and freezing blood, sperm and umbilical cord cells from roadkill and other wild animals that had died, mostly in the Cerrado savanna?an incredibly diverse collection of tropical forest and grassland ecosystems home to at least 10,000 plant species and more than 800 species of birds and mammals, some of which live nowhere else in the world. Specimens were collected from the bush dog, collared anteater, bison and gray brocket deer, among other species.
The idea was to preserve the genetic information of Brazil's endangered wildlife. One day, the organizations reasoned, they might be able to use the collected DNA to clone endangered animals and bolster dwindling populations. So far the two institutions have collected at least 420 tissue samples. Now they are collaborating on a related project that will use the DNA in these specimens to improve breeding and cloning techniques. Current cloning techniques have an average success rate of less than 5 percent, even when working with familiar species; cloning wild animals is usually less than 1 percent successful.
Any animals born during Brazil's new undertaking will live in the Brasilia Zoo, says Embrapa researcher Carlos Martins. Expanding captive populations of wild animals, he and his team hope, will discourage zoos and researchers from taking even more wild animals out of their native habitats. Martins and his colleagues have not yet decided which species they will attempt to clone but the maned wolf and jaguar are strong candidates. The International Union for Conservation of Nature classifies both animals as "near threatened" on its Red List of Threatened Species, two levels below "endangered."
Many researchers agree that, at present, cloning is not a feasible or effective conservation strategy. First of all, some conservationists point out, cloning does not address the reasons that many animals become endangered in the first place?namely, hunting and habitat destruction. Even if cloning could theoretically help in truly desperate situations, current cloning techniques are simply too ineffective to make much of a difference. Compared with cloning domestic species?particularly cattle, which have been successfully cloned for years to duplicate desirable traits?cloning endangered species is far more difficult for a number of reasons.
Successful cloning generally involves at least three essential components: DNA from the animal to be cloned; a viable egg to receive that DNA; and a mother to gestate the resulting embryo. Often, hundreds of embryos and attempted pregnancies are needed to produce even a few clones. Scientists usually have a poor understanding of endangered animals' reproductive physiology, which makes it too risky to extract a sufficient number of eggs from that species or rely on females of that species to give birth to clones. Legal protections sometimes preclude threatened species from such procedures as well. To compensate, researchers fuse the DNA of an endangered species with eggs from a closely related species and select mothers from the latter. Such hybrid embryos often fail to develop properly.
Although they are keenly aware of these problems, Martins and his colleagues, as well as a few other scientists around the world, think that efforts to archive the genetic information of endangered wildlife are worthwhile. Some researchers remain optimistic that cloning will become a useful tool for conservation in the future. Optimists point to recent successes cloning wild mammals using closely related domestic species, improved techniques for preventing developmental abnormalities in a cloned embryo, better neonatal care for newborn clones and in vitro fertilization made possible by stem cells derived from frozen tissue.
The first clones
In the early 1950s, at the Lankenau Hospital Research Institute in Philadelphia, Robert Briggs and Thomas King successfully cloned 27 northern leopard frogs through a process known as nuclear transfer. The nucleus, often called the command center of the cell, contains most of a vertebrate's DNA?except for the DNA within bean-shaped, energy-generating organelles named mitochondria. Briggs and King emptied frog eggs of their nuclei, sucked nuclei out of cells in frog embryos and injected those nuclei into the empty eggs. Many of the eggs developed into tadpoles that were genetically identical to the embryos that had donated their nuclear DNA.
In 1958 John Gurdon, then at the University of Oxford, and colleagues cloned frogs with nuclear DNA extracted from the cells of fully formed tadpoles. Unlike embryonic cells, which are genetically flexible enough to become a variety of different tissues, a tadpole's cells are "differentiated"?that is, the patterns of genes they express have changed to fit the profile of a specific cell type: a skin, eye or heart cell, for example. Gurdon demonstrated that, when transplanted into an egg, nuclear DNA from a mature cell reverts to the more versatile state characteristic of DNA in an embryo's cells. This breakthrough encouraged scientists to try cloning far larger animals using DNA from adult cells.
In 1996 researchers in Scotland attempted to clone a female Finn-Dorset sheep. They injected nuclei extracted from her udder cells into nearly 300 empty eggs derived from Scottish blackfaces, a different sheep breed. Out of those prepared eggs, the scientists managed to create more than 30 embryos. Only five of those embryos developed into lambs after being implanted in surrogate Scottish blackfaces. And only one of those lambs survived into adulthood. The researchers named her Dolly.
Since then some biologists have repeatedly suggested that cloning could help save endangered species, especially in dire situations in which only a few dozen or a handful of animals remain. The smaller, more homogenous and more inbred a population, the more susceptible it is to a single harmful genetic mutation or disease. Clones could theoretically increase the genetic diversity of an endangered population if researchers have access to preserved DNA from many different individuals. At the very least, clones could stabilize a shrinking population. And, some researchers argue, a genetically homogenous but stable population would be better than extinction; some highly inbred groups of wild animals, such as Chillingham cattle in England, have survived just fine for hundreds of years.
One species that might benefit from cloning is the northern white rhinoceros, which is native to Africa. In 1960 the global northern white rhino population was more than 2,000 strong, but poaching has reduced their numbers to as few as 11 today. By last count, three live in zoos?two in San Diego and one in the Czech Republic?four live in the Ol Pejeta Conservancy in Kenya and as few as four individuals may still live in the wild based on unconfirmed reports, but they have not been spotted in several years. Most of the captive animals are uninterested in mating or infertile, although two rhinos mated in the summer of 2012.
Right now, though, cloning is unlikely to help the white rhino or any other threatened species. To date, the story of cloning endangered animals is one of a few high-profile successes and many, many failures. Since the early 2000s, using the same technique that produced Dolly, researchers have cloned several endangered and even extinct mammals, including a mouflon sheep and a bovine known as a gaur in 2001; a kind of wild cattle called a banteng in 2003; a wild goat known as the Pyrenean ibex in 2009; and wild coyotes in 2012. In each case many more clones died before birth than survived; in most cases none of the clones survived into adulthood.
Mismatched
All those attempted clones of endangered or extinct animals died in different ways for different reasons, but they all shared one fundamental problem?they were not exact replicas of their counterparts. In most cases, researchers have combined DNA from the threatened species with eggs from a related domestic species. Each surrogate mother is often implanted with dozens of hybrid embryos in order to achieve at least a few pregnancies, a strategy that requires extracting hundreds of eggs. Because the reproductive physiology of most endangered animals is so poorly understood, researchers are often unsure when the animals ovulate and how best to acquire their eggs. In some cases legal protections prevent scientists from harvesting eggs from threatened species. For all these reasons, they turn to more familiar domestic species instead.
Injecting the DNA of one species into the egg of another species?even a closely related one?creates an unusual hybrid embryo that often fails to develop properly in the womb of a surrogate mother. Hybrid embryos have the nuclear DNA of the cloned species and the mitochondrial (mtDNA) DNA of the donor egg. This mismatch becomes problematic as the embryo develops. Nuclear DNA and mtDNA work together; they both contain genetic recipes for proteins with which cells extract energy from food. In a hybrid embryo these proteins do not always fit together properly, which leaves cells starved for energy. Complicating matters further, the surrogate mother often rejects the hybrid embryo because she recognizes some of the embryo's tissues, particularly the placenta, as foreign.
Another problem?and the most intractable so far?is that a hybrid embryo created via nuclear transfer is not a genetic blank slate like most embryos. All vertebrates begin life as hollow balls of embryonic stem cells, which can become almost any type of adult cell. Each of those stem cells contains a copy of the exact same genome packaged into chromosomes?tight bundles of DNA and histone proteins. As the embryo develops, the stem cells begin to take on their adult forms: some become skin cells, others heart cells and so on. Different types of cells begin to express different patterns of genes. Inside each cell an assortment of molecules and enzymes interacts with DNA and histones to change gene expression. Some molecules, such as methyl groups, physically block cellular machinery from reading the genetic instructions in certain segments of DNA; some enzymes loosen the bonds between histones and DNA, making particular genes more accessible. Eventually, each cell type?skin cell, liver cell, brain cell?has the same genome, but a different epigenome: a unique pattern of genes that are actively expressed or effectively silenced. Over time, an adult cell's epigenome can change even further, depending on the animal's life experiences.
So when researchers inject an adult cell's nucleus into an empty egg, the nucleus brings its unique epigenome with it. As Gurdon's early experiments in the 1950s and subsequent studies have shown, an egg is capable of erasing the epigenome of introduced nuclear DNA, wiping the slate clean?to some extent. This process of "nuclear reprogramming" is poorly understood, and the egg often fails to complete it properly, especially when the egg is from one species and the nuclear DNA from another. Incomplete nuclear reprogramming is one of the main reasons, scientists think, for the many developmental abnormalities that kill clones before birth and for the medical issues common to many survivors, such as extremely high birth weight and organ failure.
Some researchers see ways around these problems. Pasqualino Loi of the University of Teramo in Italy was part of a team that successfully cloned endangered mouflon sheep in the early 2000s; the clones died within six months of birth. Loi and his colleagues think they can increase the chances of a hybrid embryo surviving in a surrogate mother's womb. First, they propose, researchers could nurture a hybrid embryo for a short time in the lab until it develops into what is known as a blastocyst?the ball-shaped beginnings of a vertebrate composed of an outer circle of cells, the trophoblast, surrounding a clump of rapidly dividing stem cells known as the inner cell mass. Eventually, the trophoblast becomes the placenta. Researchers could scoop out the inner cell mass from the hybrid blastocyst, Loi suggests, and transplant it into an empty trophoblast derived from the same species as the surrogate mother. Because the surrogate mother is far less likely to reject a trophoblast from her own species, the developing embryo within has a much better chance of surviving.
Scientists have also figured out how to encourage nuclear reprogramming by bathing the egg in certain compounds and chemicals, such as trichostatin A, which stimulate or inhibit the enzymes that determine a cell's epigenome. Most recently, Teruhiko Wakayama of?the RIKEN Center for Developmental Biology in Kobe, Japan and his colleagues produced 581 cloned mice from a single donor mouse over 25 generations, using trichostatin A to achieve success rates as high as 25 percent in some but not all generations. To solve the mismatch of mtDNA and nuclear DNA, Loi suggests simply removing the egg's native mtDNA and replacing it with mtDNA from the species to be cloned?something that researchers tried in the 1970s and '80s, but have not attempted recently for reasons that are unclear.
Some of the most successful attempts to clone endangered animals in recent years have involved two of the most beloved domestic species?cats and dogs. At the Audubon Center for Research of Endangered Species in New Orleans, Martha Gomez and her colleagues have created many African wildcat clones since the mid-2000s, using domestic cats as surrogate mothers. Gomez says eight clones have survived into adulthood so far and are all healthy today. She attributes her success, in part, to the fact that wildcats and domestic cats are much more closely related to each other than are most wild and domestic species paired for the purpose of cloning. She and her team have also learned to increase success rates with caesarian sections?to spare clones the stress of a typical birth?and to keep newborn clones in intensive care for a few weeks, as though they were premature babies. In 2008, B. C. Lee of Seoul National University in Korea and his colleagues achieved similar success using domestic dogs to create three healthy male gray wolf clones. Lee's team had previously created two female gray wolf clones. All five animals survived into adulthood, Lee confirms.
Working with black-footed cats, which are native to Africa and listed as "Vulnerable" on the Red List, Gomez is now focusing on a method of cloning that differs from nuclear transfer. She is trying to transform adult cells from black-footed cats into stem cells and subsequently induce those stem cells to become sperm and eggs. Then, through in vitro fertilization or similar techniques, she could impregnate domestic cats with black-footed cat embryos. Alternatively, stem cell-derived sperm and eggs could be used to impregnate females of the endangered species.
To say that this approach is technically challenging would be an understatement, but researchers have made impressive progress. In 2011 Jeanne Loring of the Scripps Research Institute in La Jolla, Calif., and her colleagues produced stem cells from the frozen skin cells of two endangered species?the northern white rhino and a baboonlike primate known as a drill. And in 2012 Katsuhiko Hayashi of Kyoto University Graduate School of Medicine and colleagues turned skin cells from adult mice into stem cells, which they then transformed into viable eggs. After fertilizing the eggs with sperm in test tubes, the researchers implanted the embryos in surrogate mother mice that gave birth to healthy and fertile offspring. ?
"I'm not saying cloning is going to save endangered species," Gomez says, "but I am still a believer of cloning as another tool. It's not easy, though. The research moves slow."
Teramo?s Loi remains optimistic too. He thinks that scientists should continue to collect and preserve the genetic information of endangered animals, as Brazil has done, creating bio-banks of tissue on ice, such as the "frozen zoo" at the San Diego Zoo?s Institute for Conservation Research. If researchers manage to dramatically increase the efficiency of cloning wild and endangered animals?whether with nuclear transfer or in vitro fertilization?then the DNA they need will be waiting for them. If they do not, bio-banks will still be useful for more basic research. "Once cloning of endangered animals is properly established, it will be a very powerful tool," Loi says. "If something can be done, it will be done in 10 years."
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