n 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 homelands of India, In- Idochina and southeast Asia. These one-ton bovines have been hunted for sport for generations. More recently the gaur’s habitats of forests, bamboo jungles and grasslands have dwindled to the point that only roughly 36,000 are thought to remain in the wild. The World Conservation

Union–IUCN 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 Interna- tional Trade in Endangered Species (CITES).

But if all goes as predicted, in a few weeks a spindly-legged little Noah will trot in a new day in the conservation of his kind as well as in the preservation of many other endangered species. Perhaps most important, he will be living, mooing proof that one animal can carry and give birth to the exact genetic du- plicate, or clone, of an animal of a different species. And Noah will be just the 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 . Cloning could also reincarnate some spe- cies 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 cryo- preserve semen; similarly, eggs are difficult to obtain and are damaged by freez- ing. But by cloning animals whose body cells have been preserved, scientists can keep the genes of that individual alive, maintaining (and in some instances in- creasing) 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 fertiliza- tion, 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

SLIM FILMS (illustration); LYNDA RICHARDSON Corbis (orangutan); MARTIN WENDLER Peter Arnold, Inc. (ocelot); GERRY ELLIS Minden Pictures (panda); KENNETH W. FINK Photo Researchers, Inc. (bongo); FRANS LANTING Minden Pictures (cheetah); ROLAND SEITRE Peter Arnold, Inc. (gaur); JOHN CANCALOSI Peter Arnold, Inc. (goat) CloningCloning Noah’sNoah’s ArkArk Biotechnology might offer the best way to keep some endangered species from disappearing from the planet

by Robert P. Lanza, Betsy L. Dresser and Philip Damiani would advocate rounding up wild fe- Advanced Cell Technology (ACT) in we are now working to understand. male endangered animals for that pur- Worcester, Mass., had to fuse skin cells They are also a function of the vagaries pose or subjecting a precious zoo resi- taken from a male gaur with 692 enu- of assisted reproduction technology. dent of the same species to the rigors of cleated cow eggs. As we report in the Accordingly, we expect that the first assisted reproduction and surrogate current issue of the journal Cloning, of few endangered species to be cloned motherhood. That means that to clone those 692 cloned early embryos, only will be those whose reproduction has an endangered species, researchers such 81 grew in the laboratory into blasto- already been well studied. Several zoos as ourselves must solve the problem of cysts, balls of 100 or so cells that are and conservation societies—including how to get cells from two different sufficiently developed to implant for the Audubon Institute Center for Re- species to yield the clone of one. gestation. We ended up inserting 42 search of Endangered Species (AICRES) blastocysts into 32 cows, but only eight in New Orleans, which is led by one of A Gaur Is Born became pregnant. We removed the fe- us (Dresser)—have probed the repro- tuses from two of the pregnant cows for ductive biology of a range of endan- t is a deceptively simple-looking pro- scientific analysis; four other animals gered species, with some notable suc- Icess. A needle jabs through the protec- experienced spontaneous abortions in cesses. Last November, for example, tive layer, or zona pellucida, surrounding the second or third month of the usual Dresser and her colleagues reported the an egg that hours ago resided in a living nine-month pregnancy; and the seventh first transplantation of a previously ovary. In one deft movement, a research cow had a very unexpected late-term frozen embryo of an endangered animal assistant uses it to suck out the egg’s nu- spontaneous abortion in August. into another species that resulted in a cleus—which contains the majority of a The statistics of the efficiency of clon- live birth. In this case, an ordinary house cell’s genetic material—leaving behind ing reflect the fact that the technology is cat gave birth to an African wildcat, a

THE NUCLEAR TRANSFER (CLONING) PROCESS

ZONA PELLUCIDA PIPETTE NEEDLE (protective layer) CHROMOSOME POLAR BODY

EGG

Recipient eggs are coaxed to The polar bodies and chro- Once the chromosomes and Skin cells called fibroblasts An entire sk mature in a culture dish. mosomes of each egg are polar body are removed, all are isolated from the animal up into the n Each has a remnant egg cell drawn into a needle. A pip- that remains inside the zona to be cloned and grown in again punch called the polar body. ette holds the egg still. pellucida is cytoplasm. culture dishes. zona pellucid

only a sac of gel called cytoplasm. Next still as much an art as it is a science— species that has declined in some areas. he uses a second needle to inject anoth- particularly when it involves transplant- So far, beyond the African wildcat er, whole cell under the egg’s outer layer. ing an embryo into another species. Sci- and the gaur, we and others have ac- With the flip of an electric switch, the entists, including those of us at ACT, complished interspecies embryo trans-

cloning is complete: the electrical pulse have had the highest success rates clon- fers in four additional cases: an Indian ) fuses the introduced cell to the egg, and ing domestic cattle implanted into cows desert cat into a domestic cat; a bongo the early embryo begins to divide. In a of the same species. But even in this in- antelope into a more common African photographs few days, it will become a mass of cells stance we have had to work hard to antelope called an eland; a mouflon large enough to implant into the uterus produce just a few animals. For every sheep into a domestic sheep; and a rare of a surrogate-mother animal previous- 100 cow eggs we fuse with adult cattle red deer into a common white-tailed

ly treated with hormones. In a matter of cells, we can expect only between 15 and deer. All yielded live births. We hope ); PHILIP DAMIANI ( months, that surrogate mother will give 20 to produce blastocysts. And only that the studies of felines will pave the

birth to a clone. roughly 10 percent of those—one or way for cloning the cheetah, of which illustrations In practice, though, this technique— two—yield live births. only roughly 12,000 remain in south- which scientists call nuclear transfer— The numbers reflect difficulties with ern Africa. The prolonged courtship be-

is not so easy. To create Noah, we at the nuclear transfer process itself, which havior of cheetahs requires substantial ( GRACE LAURIE

86 Scientific American November 2000 Cloning Noah’s Ark territory, a possible explanation for gust 1999 Dayuan Chen of the institute with AICRES and Louisiana State Uni- why the animals have bred so poorly in and his co-workers published a paper versity) and their colleagues announced zoos and yet another reason to fear in the English-language journal Science the birth of a bongo after moving very their extinction as their habitat shrinks. in China announcing that they had fused early embryos from a pregnant female panda skeletal muscle, uterus and mam- bongo to an eland surrogate mother. Panda-monium mary gland cells with the eggs of a rabbit Most of the mountain subspecies of and then coaxed the cloned cells to de- bongo—a medium-size antelope with ne of the most exciting candidates velop into blastocysts in the laboratory. vertical white stripes—live in captivity. Ofor endangered-species cloning— A rabbit, of course, is too small to According to the World Conservation the giant panda—has not yet been the serve as a surrogate mother for a giant Union–IUCN, the mountain bongo is subject of interspecies transfer experi- panda. Instead ACT and the Chinese endangered, with only 50 or so remain- ments, but it has benefited from assist- plan to turn to American black bears. ing in a small region of Kenya. In con- ed reproduction technology. Following As this issue of Scientific American goes trast, the 1999 Bongo International the well-publicized erotic fumblings of to press, ACT is finalizing plans to ob- Studbook lists nearly 550 mountain the National Zoo’s ill-fated panda pair, tain eggs from female black bears killed bongo living in zoos throughout the the late Ling-Ling and Hsing-Hsing, the during this autumn’s hunting season in world. The lowland bongo subspecies turned to artificial in- the northeastern U.S. Together with the is slightly better off: it is listed as “near semination to make proud parents of Chinese, ACT scientists hope to use threatened” and has a population of its and . Baby these eggs and frozen cells from the late perhaps several thousand scattered was born in August 1999. Hsing-Hsing or Ling-Ling to generate throughout central and western Africa. Giant pandas are such emblems of en- cloned giant panda embryos that can be A coalition of conservation organiza-

NUCLEUS SKIN CELL SKIN CELL

NUCLEUS

kin cell is taken The skin cell is injected un- The process is repeated Each injected egg is exposed The skin cell’s nucleus, with needle, which is derneath the zona pellucida, many times; it can require to an electric shock that fus- its genes, enters the egg cyto- hed through the where it remains separate hundreds of nuclear trans- es the skin cell with the egg plasm. Within a few hours, da. from the egg cytoplasm. fers to obtain one animal. cytoplasm. the fused cell begins to divide.

dangered species that the World Wild- implanted into a female black bear now tions in the U.S. and Kenya is now plan- life Fund (WWF) uses one in its logo. living in a zoo. A research group that in- ning to send mountain bongo that have According to a census that is now al- cludes veterinarians at Bear Country been bred in captivity to two sites in most 20 years old, fewer than 1,000 U.S.A. in Rapid City, S.D., has already Kenya. And in a new approach to rein- pandas remain in their mountainous demonstrated that black bears can give troducing a species, AICRES is working habitats of bamboo forest in southwest birth to transplanted embryos. They re- in Kenya to transfer frozen bongo em- China. But some biologists think that ported the successful birth of a black bryos into eland surrogates. Cloning the population might have rebounded a bear cub from an embryo transferred could support these efforts and possibly bit in some areas. The WWF expects to from one pregnant black bear to another yield more bongo for reintroduction. complete a census of China’s pandas in last year in the journal Theriogenology. But what about animals that are al- mid-2002 to produce a better estimate. AICRES scientists hope to take ad- ready extinct? Chances are slim to nil In the meantime, we at ACT are dis- vantage of the success with bongo ante- that scientists will soon be able to clone cussing plans with the government of lope that one of us (Dresser) had while dinosaurs, à la Jurassic Park, or woolly China to clone a giant panda. Chinese at the Cincinnati Zoo. In 1984 Dresser mammoths. The primary problem is the scientists have already made strides to- and Charles Earle Pope of the Universi- dearth of preserved tissue—and hence ward the goal of panda cloning. In Au- ty of Alabama at Birmingham (now DNA. A group of researchers unearthed

www.sciam.com Scientific American November 2000 87 WHAT ABOUT ROVER AND FLUFFY? he list of domesticated animals that scientists have species. We expect to be able to apply the information we ob- been able to clone so far includes sheep, cattle, goats tain from cloning cats and dogs to preserving endangered fe- Tand laboratory mice—and now, we expect, the gaur. lines and canines. Compared with that menagerie, you’d think that cloning an ACT and several other companies now offer pet cloning kits ordinary dog or cat would be a snap. Unfortunately, this has that veterinarians can use to preserve samples from a client’s not been the case. Both of our research groups have created pet for possible future cloning. The kits contain materials for cloned cat embryos and have implanted them into female collecting a skin specimen and sending it back to a laboratory. cats, but as this article goes to press, neither of our teams has Research assistants there use the tissue to establish a collection yet obtained a full-term pregnancy. Dogs of pure, dividing cells called a cell line, which have presented even more problems. will be the source of donor cells for cloning. But we anticipate success soon. At Ad- ACT extracts eggs for the cloning proce- vanced Cell Technology (ACT),we have under- dure from reproductive tracts taken from an- taken a research program that uses cloning imals that have been spayed by veterinari- technology to propagate pets as well as serv- ans. We remove the ovaries and carefully ice animals such as seeing-eye dogs for the puncture all visible follicles to release the blind, hearing dogs for the deaf, search-and- eggs. Then we collect the eggs and place rescue dogs,and animals used for social thera- them in a specialized maturation medium py. Together with Louisiana State University, that contains hormones, proteins and nutri- the Audubon Institute has teamed up with a ents. Once fully matured, the eggs are ready company called Lazaron BioTechnologies in for the nuclear transfer procedure [see illus- Baton Rouge,La.,to clone pet dogs and cats. tration on pages 86 and 87]. A surprising number of people are interest- So far our main focus has been the domes- ed in cloning their favorite deceased pet in tic cat, primarily because its reproductive the hope of getting an animal with similar be- physiology has been well studied, and em- ) bottom

havioral characteristics.A good deal of a cat or bryo transfers of early- and late-stage em- ( dog’s demeanor is thought to be genetically bryos have resulted in the birth of live kit- determined. Although one can argue that SERVICE ANIMALS and pets tens. Both ACT and the Audubon Institute there are already plenty of cats and dogs in might soon be cloned. In a new have been able to establish systems for Phoenix Pictures the world that need homes, people still use film, The 6th Day, grieving pet prompting cat eggs to mature in the lab and

owners go to a company called EWAN traditional breeding methods to try to repro- have consistently produced cloned embryos C RePet to copy their animals. duce a particularly desirable animal. Cloning that are being transferred to recipients. ); ROB M top

could offer a more efficient alternative.It could be particularly But dogs are a different story. The dog’s reproductive physi- ( important in the case of service animals.Currently,for instance, ology is unique among mammalian species. Dogs ovulate an male seeing-eye dogs are neutered at an early age so that they immature egg that has a very long maturation time. This can concentrate better during their expensive and rigorous means that we need a different maturation system from the training. So, unfortunately, even if a dog turns out to be very one we have used in cats and that we have fewer eggs to work BIOS Peter Arnold,BIOS Peter Inc. good at his job,he can’t be bred to produce more like him. with in the end. So Fluffy will probably have a leg up on Rover Our efforts to clone pets could also pay off for endangered when it comes to cloning. —R.P.L.,B.L.D.and P.D. KLEIN/HOBERT what they had hoped would be a well- rection. ACT is arranging a collabora- microsome to add a Y chromosome preserved mammoth last year, but re- tion with Alberto Fernández-Arias and from a closely related goat species. The peated freezing and thawing over the José Folch of the Agricultural Research technology has been used by other re- eons had poked holes in the creature’s Service in Zaragoza, Spain. Fernández- searchers to manipulate human chromo- DNA, and molecular biologists have Arias froze tissue from the last bucardo. somes, but it has never before been used not yet found a feasible way of filling in He and Folch had tried for several years for cloning. A nonprofit organization such genetic gaps. to preserve the mountain goat, which in called the Soma Foundation has been es- A similar difficulty has hobbled ef- the end was wiped out by poaching, tablished to help fund such efforts. forts by Australian scientists to clone a habitat destruction and landslides. Last thylacine, or Tasmanian tiger, a wolflike year they transferred embryos from a Why Clone? marsupial that died out in the 1930s. subspecies related to the bucardo to a Researchers at the Australian Museum domestic goat, yielding live kids. loning endangered species is contro- in Sydney are attempting to clone cells But even if interspecies nuclear trans- Cversial, but we assert that it has an from a thylacine pup that was preserved fer succeeds for the bucardo, it will yield important place in plans to manage in alcohol in 1866, but the DNA is in only a sorority of clones, because we species that are in danger of extinction. such poor condition that they say they have tissue from just one animal, a fe- Some researchers have argued against it, will have to reconstruct all of the ani- male. ACT plans to try to make a male maintaining that it would restrict an al- mal’s chromosomes. by removing one copy of the X chromo- ready dwindling amount of genetic di- The recently extinct bucardo may some from one of the female bucardo’s versity for those species. Not so. We ad- prove a more promising target for resur- cells and using a tiny artificial cell called a vocate the establishment of a worldwide

88 Scientific American November 2000 Cloning Noah’s Ark ); giant panda ( Minden Pictures ) ocelot ( ); FRANS LANTING bongo Peter Arnold,Peter Inc. ( ); MARTIN WENDLER Photo Researchers, Inc. gaur ( Peter Arnold,Peter Inc. ); CHARLES V. ANGELO cheetah ( ); ROLAND SEITRE bucardo Minden Pictures ( GERRY ELLIS GERRY Peter Arnold,Peter Inc. JOHN CANCALOSI JOHN CANCALOSI Left to right,Left clockwise: network of repositories to hold frozen cells that could be used for cloning. CLONING CANDIDATES include (clock- tissue from all the individuals of an en- Other critics claim that the practice wise from upper left) the cheetah, bongo, dangered species from which it is possi- could overshadow efforts to preserve giant panda, bucardo, gaur and ocelot. ble to collect samples. Those cells—like habitat. We counter that while habitat the sperm and eggs now being collected preservation is the keystone of species ly enterprise in the case of large animals. in “frozen zoos” by a variety of zoolog- conservation, some countries are too Another argument against cloning en- ical parks—could serve as a genetic trust poor or too unstable to support sustain- dangered species is that it might siphon for reconstituting entire populations of able conservation efforts. What is more, donor money away from habitat main- a given species. Such an enterprise the continued growth of the human tenance. But not all potential donors are would be relatively inexpensive: a typi- species will probably make it impossible willing to support efforts to stem the tide cal three-foot freezer can hold more to save enough habitat for some other of habitat destruction. We should recog- than 2,000 samples and uses just a few species. Cloning by interspecies nuclear nize that some who would otherwise not dollars of electricity per year. Currently transfer offers the possibility of keeping donate to preserve endangered species at only AICRES and the San Diego Zoo’s the genetic stock of those species on all might want to support cloning or Center for Reproduction of Endangered hand without maintaining populations other assisted reproduction technologies. Species maintain banks of frozen body in captivity, which is a particularly cost- The time to act is now. SA

The Authors Further Information

ROBERT P. LANZA, BETSY L. DRESSER and PHILIP DAMIANI Preservation of Endangered Species and Populations: share an interest in reproductive biology and animals. Lanza is vice A Role for Genome Banking, Somatic Cell Cloning, president of medical and scientific development at Advanced Cell Tech- and Androgenesis? Graham E. Corley-Smith and Bruce P. nology (ACT) in Worcester, Mass. He founded the South Meadow Brandhorst in Molecular Reproduction and Development, Pond and Wildlife Association in Worcester County and is a member of Vol. 53, No. 3, pages 363–367; July 1999. the conservation commission of Clinton Township. Dresser is senior Biodiversity Hotspots for Conservation Priorities. Nor- vice president for research at the Audubon Institute and director of the man Myers, Russell A. Mittermeier, Cristina G. Mittermeier, Audubon Institute Center for Research of Endangered Species and the Gustavo A. B. da Fonseca and Jennifer Kent in Nature, Vol. Freeport-McMoRan Audubon Species Survival Center, all in New Or- 403, No. 6772, pages 853–858; February 24, 2000. leans. Damiani, a research scientist at ACT, is also a member of the In- Vanishing before Our Eyes. E. O. Wilson in Time (special ternational Embryo Transfer Society’s committee on cryopreservation. report on Earth Day 2000), pages 29–34; April–May 2000. www.sciam.com Scientific American November 2000 89