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Cloning Noah's Ark Sci Am Nov 2000

Robert P. Lanza, Betsy L. Dresser and Philip Damiani

In late November a humble Iowa cow is slated to give birth to the world's first cloned endangered species, a baby bull to be named Noah. Noah is a gaur: a member of a species of large oxlike animals that are now rare in their A Gaur Is Born homelands of India, Indochina and southeast Asia. These one-ton bovines have been hunted for sport for generations. More recently the gaur's habitats of forests, Panda-monium bamboo jungles and grasslands have dwindled to the point that only roughly 36,000 are thought to remain in the Why Clone? wild. The World Conservation Union­IUCN Red Data Book lists the gaur as endangered, and trade in live gaur or gaur products--whether horns, hides or hooves--is banned by the Convention on International Trade in Endangered Species (CITES).

What About But if all goes as predicted, in a few weeks a Rover and spindly-legged little Noah will trot in a new day in the Fluffy? conservation of his kind as well as in the preservation of many other endangered species. Perhaps most important, he The Nuclear will be living, mooing proof that one animal can carry and Transfer give birth to the exact genetic duplicate, or clone, of an (Cloning) animal of a different species. And Noah will be just the Process first creature up the ramp of the ark of endangered species that we and other scientists are currently attempting to clone: plans are under way to clone the African bongo antelope, the Sumatran tiger and that favorite of zoo lovers, the reluctant-to-reproduce giant panda. Cloning could also reincarnate some species that are already extinct--most immediately, perhaps, the bucardo mountain goat of Spain. The last bucardo--a female--died of a smashed skull when a tree fell on it early this year, but Spanish scientists have preserved some of its cells. Advances in cloning offer a way to preserve and propagate endangered species that reproduce poorly in zoos until their habitats can be restored and they can be reintroduced to the wild. Cloning's main power, however, is that it allows researchers to introduce new genes back into the gene pool of a species that has few remaining animals. Most zoos are not equipped to collect and cryopreserve semen; similarly, eggs are difficult to obtain and are damaged by freezing. But by cloning animals whose body cells have been preserved, scientists can keep the genes of that individual alive, maintaining (and in some instances increasing) the overall genetic diversity of endangered populations of that species.

Nevertheless, some conservation biologists have been slow to recognize the benefits of basic assisted reproduction strategies, such as in vitro fertilization, and have been hesitant to consider cloning. Although we agree that every effort should be made to preserve wild spaces for the incredible diversity of life that inhabits this planet, in some cases either the battle has already been lost or its outcome looks dire. Cloning technology is not a panacea, but it offers the opportunity to save some of the species that contribute to that diversity. A clone still requires a mother, however, and very few conservationists would advocate rounding up wild female endangered animals for that purpose or subjecting a precious zoo resident of the same species to the rigors of assisted reproduction and surrogate motherhood. That means that to clone an endangered species, researchers such as ourselves must solve the problem of how to get cells from two different species to yield the clone of one.

A Gaur Is Born

It is a deceptively simple-looking process. A needle jabs through the protective layer, or zona pellucida, surrounding an egg that hours ago resided in a living ovary. In one deft movement, a research assistant uses it to suck out the egg's nucleus--which contains the majority of a cell's genetic material--leaving behind only a sac of gel called cytoplasm. Next he uses a second needle to inject another, whole cell under the egg's outer layer. With the flip of an electric switch, the cloning is complete: the electrical pulse fuses the introduced cell to the egg, and the early embryo begins to divide. In a few days, it will become a mass of cells large enough to implant into the uterus of a surrogate-mother animal previously treated with hormones. In a matter of months, that surrogate mother will give birth to a clone.

In practice, though, this technique--which scientists call nuclear transfer-- is not so easy. To create Noah, we at Advanced Cell Technology (ACT) in Worcester, Mass., had to fuse skin cells taken from a male gaur with 692 enucleated cow eggs. As we report in the current issue of the journal Cloning, of those 692 cloned early embryos, only 81 grew in the laboratory into blastocysts, balls of 100 or so cells that are sufficiently developed to implant for gestation. We ended up inserting 42 blastocysts into 32 cows, but only eight became pregnant. We removed the fetuses from two of the pregnant cows for scientific analysis; four other animals experienced spontaneous abortions in the second or third month of the usual nine-month pregnancy; and the seventh cow had a very unexpected late-term spontaneous abortion in August.

The statistics of the efficiency of cloning reflect the fact that the technology is still as much an art as it is a science--particularly when it involves transplanting an embryo into another species. Scientists, including those of us at ACT, have had the highest success rates cloning domestic cattle implanted into cows of the same species. But even in this instance we have had to work hard to produce just a few animals. For every 100 cow eggs we fuse with adult cattle cells, we can expect only between 15 and 20 to produce blastocysts. And only roughly 10 percent of those--one or two--yield live births.

The numbers reflect difficulties with the nuclear transfer process itself, which we are now working to understand. They are also a function of the vagaries of assisted reproduction technology.

Accordingly, we expect that the first few endangered species to be cloned will be those whose reproduction has already been well studied. Several zoos and conservation societies--including the Audubon Institute Center for Research of Endangered Species (AICRES) in New Orleans, which is led by one of us (Dresser)--have probed the reproductive biology of a range of endangered species, with some notable successes. Last November, for example, Dresser and her colleagues reported the first transplantation of a previously frozen embryo of an endangered animal into another species that resulted in a live birth. In this case, an ordinary house cat gave birth to an African wildcat, a species that has declined in some areas.

So far, beyond the African wildcat and the gaur, we and others have accomplished interspecies embryo transfers in four additional cases: an Indian desert cat into a domestic cat; a bongo antelope into a more common African antelope called an eland; a mouflon sheep into a domestic sheep; and a rare red deer into a common white-tailed deer. All yielded live births. We hope that the studies of felines will pave the way for cloning the cheetah, of which only roughly 12,000 remain in southern Africa. The prolonged courtship behavior of cheetahs requires substantial territory, a possible explanation for why the animals have bred so poorly in zoos and yet another reason to fear their extinction as their habitat shrinks.


One of the most exciting candidates for endangered-species cloning--the giant panda--has not yet been the subject of interspecies transfer experiments, but it has benefited from assisted reproduction technology. Following the well-publicized erotic fumblings of the National Zoo's ill-fated panda pair, the late Ling-Ling and Hsing-Hsing, the San Diego Zoo turned to artificial insemination to make proud parents of its Bai Yun and Shi Shi. Baby Hua Mei was born in August 1999.

Giant pandas are such emblems of endangered species that the World Wildlife Fund (WWF) uses one in its logo. According to a census that is now almost 20 years old, fewer than 1,000 pandas remain in their mountainous habitats of bamboo forest in southwest China. But some biologists think that the population might have rebounded a bit in some areas. The WWF expects to complete a census of China's pandas in mid-2002 to produce a better estimate.

In the meantime, we at ACT are discussing plans with the government of China to clone a giant panda. Chinese scientists have already made strides toward the goal of panda cloning. In August 1999 Dayuan Chen of the institute and his co-workers published a paper in the English-language journal Science in China announcing that they had fused panda skeletal muscle, uterus and mammary gland cells with the eggs of a rabbit and then coaxed the cloned cells to develop into blastocysts in the laboratory. [Image] A rabbit, of course, is too small to serve as a surrogate mother for a giant panda. Instead ACT and the Chinese plan to turn to American black bears. As this issue of Scientific American goes to press, ACT is finalizing plans to obtain eggs from female black bears killed during this autumn's hunting season in the northeastern U.S. Together with the Chinese, ACT scientists hope to use these eggs and frozen cells from the late Hsing-Hsing or Ling-Ling to generate cloned giant panda embryos that can be implanted into a female black bear now living in a zoo. A research group that includes veterinarians at Bear Country U.S.A. in Rapid City, S.D., has already demonstrated that black bears can give birth to transplanted embryos. They reported the successful birth of a black bear cub from an embryo transferred from one pregnant black bear to another last year in the journal Theriogenology.

AICRES scientists hope to take advantage of the success with bongo antelope that one of us (Dresser) had while at the Cincinnati Zoo. In 1984 Dresser and Charles Earle Pope of the University of Alabama at Birmingham (now with AICRES and Louisiana State University) and their colleagues announced the birth of a bongo after moving very early embryos from a pregnant female bongo to an eland surrogate mother.

Most of the mountain subspecies of bongo--a medium-size antelope with vertical white stripes--live in captivity. According to the World Conservation Union­IUCN, the mountain bongo is endangered, with only 50 or so remaining in a small region of Kenya. In contrast, the 1999 Bongo International Studbook lists nearly 550 mountain bongo living in zoos throughout the world. The lowland bongo subspecies is slightly better off: it is listed as "near threatened" and has a population of perhaps several thousand scattered throughout central and western Africa.

A coalition of conservation organizations in the U.S. and Kenya is now planning to send mountain bongo that have been bred in captivity to two sites in Kenya. And in a new approach to reintroducing a species, AICRES is working in Kenya to transfer frozen bongo embryos into eland surrogates. Cloning could support these efforts and possibly yield more bongo for reintroduction.

But what about animals that are already extinct? Chances are slim to nil that scientists will soon be able to clone dinosaurs, à la Jurassic Park, or woolly mammoths. The primary problem is the dearth of preserved tissue--and hence DNA. A group of researchers unearthed what they had hoped would be a well-preserved mammoth last year, but repeated freezing and thawing over the eons had poked holes in the creature's DNA, and molecular biologists have not yet found a feasible way of filling in such genetic gaps.

A similar difficulty has hobbled efforts by Australian scientists to clone a thylacine, or Tasmanian tiger, a wolflike marsupial that died out in the 1930s. Researchers at the Australian Museum in Sydney are attempting to clone cells from a thylacine pup that was preserved in alcohol in 1866, but the DNA is in such poor condition that they say they will have to reconstruct all of the animal's chromosomes.

The recently extinct bucardo may prove a more promising target for resurrection. ACT is arranging a collaboration with Alberto Fernández-Arias and José Folch of the Agricultural Research Service in Zaragoza, Spain. Fernández-Arias froze tissue from the last bucardo. He and Folch had tried for several years to preserve the mountain goat, which in the end was wiped out by poaching, habitat destruction and landslides. Last year they transferred embryos from a subspecies related to the bucardo to a domestic goat, yielding live kids.

But even if interspecies nuclear transfer succeeds for the bucardo, it will yield only a sorority of clones, because we have tissue from just one animal, a female. ACT plans to try to make a male by removing one copy of the X chromosome from one of the female bucardo's cells and using a tiny artificial cell called a microsome to add a Y chromosome from a closely related goat species. The technology has been used by other researchers to manipulate human chromosomes, but it has never before been used for cloning. A nonprofit organization called the Soma Foundation has been established to help fund such efforts.

Why Clone?
Cloning endangered species is controversial, but we assert that it has an important place in plans to manage species that are in danger of extinction. Some researchers have argued against it, maintaining that it would restrict an already dwindling amount of genetic diversity for those species. Not so. We advocate the establishment of a worldwide network of repositories to hold frozen tissue from all the individuals of an endangered species from which it is possible to collect samples.Those cells--like the sperm and eggs now being collected in "frozen zoos" by a variety of zoological parks--could serve as a genetic trust for reconstituting entire populations of a given species. Such an enterprise would be relatively inexpensive: a typical three-foot freezer can hold more than 2,000 samples and uses just a few dollars of electricity per year. Currently only AICRES and the San Diego Zoo's Center for Reproduction of Endangered Species maintain banks of frozen body cells that could be used for cloning.

Other critics claim that the practice could overshadow efforts to preserve habitat. We counter that while habitat preservation is the keystone of species conservation, some countries are too poor or too unstable to support sustainable conservation efforts. What is more, the continued growth of the human species will probably make it impossible to save enough habitat for some other species. Cloning by interspecies nuclear transfer offers the possibility of keeping the genetic stock of those species on hand without maintaining populations in captivity, which is a particularly costly enterprise in the case of large animals.

Another argument against cloning endangered species is that it might siphon donor money away from habitat maintenance. But not all potential donors are willing to support efforts to stem the tide of habitat destruction. We should recognize that some who would otherwise not donate to preserve endangered species at all might want to support cloning or other assisted reproduction technologies.

The time to act is now. -----------------------------------------------------------

Further Information:

PRESERVATION OF ENDANGERED SPECIES AND POPULATIONS: A ROLE FOR GENOME BANKING, SOMATIC CELL CLONING, AND ANDROGENESIS? Graham E. Corley-Smith and Bruce P. Brandhorst in Molecular Reproduction and Development, Vol. 53, No. 3, pages 363­367; July 1999.

BIODIVERSITY HOTSPOTS FOR CONSERVATION PRIORITIES. Norman Myers, Russell A. Mittermeier, Cristina G. Mittermeier, Gustavo A. B. da Fonseca and Jennifer Kent in Nature, Vol. 403, No. 6772, pages 853­858; February 24, 2000.

VANISHING BEFORE OUR EYES. E. O. Wilson in Time (special report on Earth Day 2000), pages 29­34; April­May 2000. -----------------------------------------------------------

The Authors

ROBERT P. LANZA, BETSY L. DRESSER and PHILIP DAMIANI share an interest in reproductive biology and animals. Lanza is vice president of medical and scientific development at Advanced Cell Technology (ACT) in Worcester, Mass. He founded the South Meadow Pond and Wildlife Association in Worcester County and is a member of the conservation commission of Clinton Township. Dresser is senior vice president for research at the Audubon Institute and director of the Audubon Institute Center for Research of Endangered Species and the Freeport-McMoRan Audubon Species Survival Center, all in New Orleans. Damiani, a research scientist at ACT, is also a member of the International Embryo Transfer Society's committee on cryopreservation. ------------------------------------------------------------------------