TRANSGENIC ANIMALS: Infant Monkey Carries Jellyfish Gene
by
Gretchen Vogel,
Science Writer

Close, but no "glow" yet.

[ANDi, the first transgenic Rhesus monkey, carries the gene for a green fluorescent protein derived from Jellyfish, but he doesn't glow yet, as have other mammals, like mice, who have undergone this sort of procedure.]

In an attempt to create the first transgenic primate, scientists have produced a Rhesus monkey that carries the gene coding for green fluorescent protein. Although he is not green, the monkey, described on p. 309 of the January 12th issue of Science [See Abstract below.], could lead to valuable experimental models for certain diseases and a better understanding of primate and human development, say other biologists. But the cumbersome technique is not likely to lead to transgenic humans, green or otherwise.

To produce ANDi, reproductive biologists Anthony Chan, Gerald Schatten, and colleagues injected a genetically modified virus into the unfertilized eggs of rhesus monkeys. A few hours later, they injected sperm into the oocytes to fertilize them. As with other in vitro fertilization (IVF) procedures in nonhuman primates, this one was relatively inefficient. Half of the fertilized eggs developed into embryos, and five pregnancies resulted from 20 embryo transfers, including one set of twins, which were miscarried. [Editor's Note: This is not unusual in monkeys with twins.]

Three healthy monkeys were born, but the team has detected the GFP gene only in ANDi. The miscarried twins also carried the GFP gene, but unlike ANDi, their hair follicles and toenails did glow under fluorescent light. Schatten attributes the miscarriage to the fact that Rhesus twins are rare, but the team is investigating whether it might be related to the inserted gene. So far, the team doesn't know whether ANDi's cells are expressing the protein. But Schatten says other transgenic animals have delayed producing their transgene for up to a year after birth.

Although the gene transfer techniques the researchers used are routine in other organisms, reproductive biologist Ted Golos of the Wisconsin Regional Primate Research Center in Madison says "the birth of ANDi is the first demonstration that a primate egg can develop normally after such manipulations."

"We've made an incremental step from one species to another," Schatten says. And even that small step involved multiple hurdles. Whereas the experiment "is essentially several days' work in transgenic mice," Golos notes, monkey eggs are difficult to collect, and primatologists do not know how to artificially control a monkey's reproductive cycle. That meant the researchers had to time the experiment precisely so that an embryo was ready when a surrogate mother was at the right stage of her reproductive cycle. In fact, ethical considerations aside, the project might have been easier to achieve in humans, for whom IVF technology is much more advanced.

"Even so, the work will not inspire fertility doctors to try the technique with human embryos anytime soon," Schatten predicts. Scientists can't control where the modified virus enters the genome, so the risk of an inserted gene interrupting an important gene would be relatively high. "I don't see an immediate therapeutic application," says bioethicist LeRoy Walters of the Kennedy Institute of Ethics at Georgetown University.

And until researchers find more efficient ways to create specific genetic changes, says Schatten, transgenic monkeys will not be common research tools. Even if those techniques were feasible, expense and ethical considerations would limit the use of transgenic monkeys as medical models, he says: "We don't need a knockout monkey for every disease."

"But for questions that are difficult to study in rodents, such as those related to aging, neurodegenerative diseases, immunology, and behavior, transgenic primates could prove a plus," Golos says. Schatten predicts that "genetically altered monkeys could be a boon to developmental biologists as well." Because monkeys are large enough to fit into magnetic resonance imaging machines, researchers might be able to introduce gene markers and track organ development by noninvasive means. "ANDi and his future cousins and brothers and sisters will help us bridge that gap between what we know in the mouse and what we're keenly interested in in human development," he says.

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A. W. S. Chan, K. Y. Chong, C. Martinovich, C. Simerly, G. Schatten*, "Transgenic Monkeys Produced by Retroviral Gene Transfer into Mature Oocytes," Science, Vol. 291, pp. 309-312 (January 12, 2001).

ABSTRACT:

Transgenic rhesus monkeys carrying the green fluorescent protein (GFP) gene were produced by injecting pseudotyped replication-defective retroviral vector into the perivitelline space of 224 mature rhesus oocytes, later fertilized by intracytoplasmic sperm injection. Of the three males born from 20 embryo transfers, one was transgenic when accessible tissues were assayed for transgene DNA and messenger RNA. All tissues that were studied from a fraternal set of twins, miscarried at 73 days, carried the transgene, as confirmed by Southern analyses, and the GFP transgene reporter was detected by both direct and indirect fluorescence imaging.

Oregon Regional Primate Research Center, Center for Women's Health, and Departments of Cell-Developmental Biology and Obstetrics-Gynecology, Oregon Health Sciences University, 505 NW 185th Avenue, Beaverton, OR 97006, USA.

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* To whom correspondence should be addressed. E-mail: schatten@ohsu.edu.

ANDi, a Genetically Modified Rhesus Monkey
by
Elizabeth Cohen,
Medical Correspondent

January 11, 2001; Portland, OR (CNN) -- A genetically modified monkey could be the key to one day curing a number of human diseases, researchers said Thursday. The baby Rhesus monkey -- named ANDi, for "inserted DNA" spelled backwards -- carries in him an extra bit of DNA from a jellyfish. Although mice have been altered in this way for years, ANDi is the first primate to be similarly modified.

Researchers at the Oregon Regional Primate Research Center (ORPRC) said their technique could eventually be used to insert a human disease gene into a monkey, creating a better way of studying diseases like Alzheimer's, diabetes, and breast cancer. The center is part of the Oregon Health Sciences University (OHSU).

"In order to eradicate these diseases we need to have the relevant disease models to perfect these innovative cures and make sure they are safe and optimized before testing them on patients," said Dr. Gerald Schatten, who led the research at the primate center. Because monkeys are genetically closer to humans than mice, they could give scientists a better understanding of how diseases develop in people. The research was described in Friday's issue of the journal Science.

Modifying a Monkey

To create ANDi, Schatten and his team injected the jellyfish gene for green fluorescence into the unfertilized egg of a normal female Rhesus monkey. The jellyfish DNA joined with the monkey DNA. The egg was then fertilized with sperm from a normal male monkey. Three monkeys were successfully altered in this way, but only ANDi survived.

"The jellyfish gene was used," Schatten said, "because it is known to be harmless and because it is easily detectable." ANDi appears normal so far -- he does not glow the way a jellyfish does. But the two other monkeys who got the gene did exhibit fluorescence. Their hair and fingernails glowed green when exposed to ultraviolet light under a microscope.

Eventually, Schatten said, scientists hope to insert other types of harmless genetic markers that can be tracked with magnetic resonance or PET scans. "If successful, doctors might be able to monitor the developmental events that lead to many diseases," he added.

Animal Rights Concerns

Animal rights activists were critical of the new research. "Meddling with the building blocks of life is extremely dangerous," said Peter Wood, a spokesman for People for the Ethical Treatment of Animals (PETA). "It goes right to the core of the research philosophy, which is 'I can do with animals as I please. I can even change their physiology.'" Wood also said he "doubted the work would yield new discoveries to treat human diseases." But Dr. John Strandberg, Director of Comparative Medicine at the National Institutes of Health, disagreed. He said "monkeys could prove to be very useful in studying human diseases."

"The genetically modified animals in the lab have been principally mice, which are good but far removed from humans. How relevant their results are to humans is questionable," he said. "The closer you can get to the human situation, the more accurate the results will be."

Next Step -- Humans?

So now that scientists have put a jellyfish gene into a monkey, do they now want to insert a gene from a non-human animal into a human being? No, said Schatten. "We don't support any extension or extrapolation of this work from laboratory animals to humans."

Several scientists and ethicists interviewed by CNN said they don't know of anyone who's interested in inserting animal genes into humans because it could be risky and has no known medical use But bioethicist Prof. Arthur Caplan at the University of Pennsylvania said "even though no one's interested now, the work with ANDi is a baby step to genetically engineering ourselves; we still have a long way to go." "The time for the public to discuss the ethics of genetic engineering is now, even though we're just at the start of the genetic trail," he added.

Update: Gene-Altered Primate Described
by
William McCall,
Associated Press Writer

January 12, 2001; Portland, OR (AP) -- Pushing science to the brink of altering humans, researchers have created the world's first genetically modified primate -- a baby rhesus monkey with jellyfish DNA that glows green in the dark. The Oregon Health Science University researchers who created ANDi -- for "inserted DNA," spelled backward -- said their goal is not to tinker with the human blueprint but to use monkeys in the laboratory to advance medical research and wipe out diseases.

The researchers hope to introduce other genes in rhesus monkeys that could trigger diseases like Alzheimer's, diabetes, breast cancer, or HIV. Then, those monkeys could be used in experiments aimed at blocking diseases at the genetic level. "I think we're at an extraordinary moment in the history of humans," said Dr. Gerald Schatten, who is leading the research at the Oregon Regional Primate Center. Mice have been genetically modified in labs and used for medical research for decades, but ANDi proves that scientists can now successfully tinker with the chromosomes of a close genetic cousin to man.

"This is a step in the direction of working with an animal that is closer in biology to humans,'' said Dr. Phyllis Leppert at the National Institutes of Health, which funded the research. She said mice have their limits when scientists try to apply experimental results to humans. Rhesus monkeys, however, share roughly 95 percent of their genes with humans, she noted. ANDi's creation, detailed in Friday's issue of the journal Science, drew cautious and critical responses from scientists around the world. "Although medical benefits may result from producing genetically modified monkeys, this sort of work must be subject to stringent monitoring," said Patrick Bateson, chairman of a Royal Society Committee on Genetically Modified Animals in Britain, where Dolly the sheep made international headlines after she was cloned in 1996.

Ray Greek, spokesman for the Physicians Committee for Responsible Medicine, questioned whether research using genetically modified monkeys would someday protect humans from cancer and heart disease. "The odds are astronomically against it," he said. "We have been doing to mice for [20 - 30] years what they have done with ANDi, and we have been singularly unsuccessful, especially in cancer research."

A year ago, Schatten reported the first monkey successfully cloned by embryo splitting. ANDi was created the same way, but he received an extra gene while he was still an unfertilized egg. The gene is a harmless protein taken from jellyfish that gives off a fluorescent green light that can be seen only under a microscope. Schatten and other researchers modified and then fertilized more than 200 rhesus monkey eggs. Forty embryos were produced and resulted in five pregnancies and three live births. Of the three baby monkeys, only ANDi proved to have the modified jellyfish gene.

He was born in October. Except for the telltale genetic marker, the monkey is normal and healthy. Greek warned that monkeys are different enough from humans that comparisons can be as difficult -- or as useless -- as comparisons with mice. Schatten countered that modifying monkey DNA eliminates the need to breed a large pool of test animals to find desired characteristics.

The research also raises questions about the ethics of making animals diseased so that they can be used in medical experiments. But Schatten said, "I don't think any of us would want to make primates sick unless it would truly accelerate the day that diseases might be eradicated."

Terri Lomax, an Oregon State University plant geneticist, said the ethics and safety of genetically modifying organisms are going to get more and more attention. "But I think people will be a little more fearful because monkeys are a little closer to home," she said.

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On the Web:
1. Oregon Health Sciences University: www.ohsu.edu;
2. Physicians Committee for Responsible Medicine: www.pcrm.org; and
3. The Royal Society: www.royalsoc.ac.uk.

Genetically Modified Monkey Created

January 11, 2001; Westport, CT (Reuters Health) -- Researchers from the Oregon Health Sciences University have created the world's first genetically modified nonhuman primate. This achievement has implications for the future study and treatment of many human genetic disorders, they state in the January 12th issue of Science.

Dr. Gerald Schatten and colleagues used a neutralized retroviral vector to introduce the green fluorescent protein (GFP) gene into 224 mature rhesus monkey oocytes. These oocytes were then fertilized by intracytoplasmic sperm injection.

Twenty embryo transfers were performed that led to five pregnancies, the authors state. From these pregnancies, three healthy male monkeys were born, and one of them, named ANDi ("inserted DNA" spelled backward), demonstrated GFP gene DNA and mRNA in all accessible tissues. In addition, the transgene was present in all tissues assayed from the two stillborn monkeys. "ANDi's birth along with last year's birth of Tetra, the first rhesus cloned by embryo splitting, is another incremental step in accelerating the discovery of innovative cures for devastating diseases," Dr. Schatten commented in a university press release.

"Nonhuman primates are invaluable models for advancing gene therapy treatments for diseases such as Parkinson's and diabetes, as well as ideal models for testing cell therapies and vaccines, including those for HIV," the authors point out. "Although we have demonstrated transgene introduction in rhesus monkeys, significant hurdles remain for the successful homologous recombination essential for gene targeting." Dr. Schatten's team believes that "the molecular approaches for making clones [by] utilizing stem cells, and now producing transgenic monkeys, could be combined to produce the ideal models to accelerate discoveries and to bridge the scientific gap between transgenic mice and humans."

Editorial Remark: The yield on this procedure is as terrible as it was with Dolly the sheep, and we better figure out what's going on if we're going to scale up to reliable industrial-strength procedures. First, Oregon researchers started with 224 oocytes (a non- trivial collection task). But only 40 embryos were obtained with the transgenic gene (less than 18 percent yield using this particular retrovirus vector). Then there are the abysmal statistics associated with IVF implantation [The 70 percent failure rate of IVF treatments for human infertility is now believed to be secondary to chromosomal aberrations, ranging from multiple aneuploidy to partial deletions and their combinations]. Twenty surrogate mothers received two embryos each, from which only five pregnancies resulted (25 percent yield). It's really tough to get each surrogate in a receptive state, since there's no way to synchronize the cycling in these females according to the convenience of the experimenter. Finally, there were only three live births (60 percent yield); the remaining pregnancies suffered spontaneous abortions, I guess. The experimenters then had the misfortune to have twins in one of the surrogate mothers, which, of course, led to a miscarriage of both twins at birth (Sigh!) [Did they do a C-section, as obstetricians are likely to do for human multiparous mothers?]. Therefore, of the three remaining singletons, only one survived.

If one multiplies the yields throughout the above equation: 0.18 x 0.25 x 0.60 x 0.33 = 0.009, one gets less than a 1 percent chance of getting a positive result. From an engineering point of view, if the truth be told, these odds are pathetic, and we had better get our act together in terms of technique. The sad story of the death of the gaur clone that was just announced today is consistent with these bad statistics. The sad death of an entire litter of cloned piglets tells a similar story. Problems with primiparous gilts during their first litter with heightened attention during the farrowing process meant that the sow did not produce the milk that was needed this time around, and for some reason the piglets did not respond favorably to supplemental milk. [Ref. Michael D. Bishop, President, Infigen, Deforest, WI, mbishop@infigen.com, "Cloned Pig Litter Update," Nature Biotechnology, Vol. 18, No. 12, p. 1227 (December 2000).