Mammalian Cloning Milestone: Mice from Mice from Mice

It was fitting, perhaps, that Cumulina the cloned mouse made her debut at a press conference in New York City on Gregor Mendel's birthday, July 22. As the father of genetics, Mendel explained genetic variability. As the first mouse cloned from an adult's cell nucleus, Cumulina represents the ultimate in genetic uniformity. So far, 50 mice have been cloned, some through three generations. Photo: ProBio America Inc. THREE GENERATIONS: Researchers at the University of Hawaii cloned these three generations of mice. At the top is the original nucleus donor. The middle and bottom levels show the second and third generations. The cloning experiments began last summer in the laboratory of Ryuzo Yanagimachi, a professor of anatomy and reproductive biology at the John A. Burns School of Medicine at the University of Hawaii. Postdoctoral research associate Teruhiko Wakayama led the project, and other team members were postdoctoral researchers Anthony Perry and Maurizio Zuccotti , and Kenneth Johnson, a research scientist from the Jackson Laboratory in Bar Harbor, Maine. The work, reported in the July 23 Nature (T. Wakayama et al. Nature, 394:369-74, July 23, 1998) was supported by the National Institutes of Health and ProBio America Inc. of Honolulu. The long-sought cloning of Mus musculus, a favorite model organism, has galvanized the agricultural, developmental biology, and biotechnology communities. "We had heard rumors about these papers for six months, and I was one of the most active skeptics. One problem I had with the work was that I didn't do it! It is an absolutely beautiful paper, with a sufficient number of mice and experiments to draw conclusions," says James Robl, a professor of veterinary and animal sciences at the University of Massachusetts in Amherst and co-founder of Advanced Cell Technology Inc. in Shrewsbury, Mass., which is developing embryonic stem (ES) cell and cloning technologies in cows. (ES cells can specialize into any cell type. See R. Lewis, The Scientist, 11[19]:1, Sept. 29, 1997). Adds Robert Wall, a research physiologist at the U.S. Department of Agriculture in Beltsville, Md.: "This work provides a new tool to answer old questions, and new questions can be posed. It is a win-win situation for the scientific community." The "Honolulu technique" for cloning mice extends the work on Dolly, which some questioned because her donor nucleus may have not been fully mature (I. Wilmut et al., Nature, 385:810-13, 1997). The July 23, 1998 Nature also includes two convincing comparisons of the famous sheep's DNA to that of her donor (D. Ashworth et al., Nature, 394:329, July 23, 1998, and E.N. Signer et al, p. 329). " Alec Jeffreys (of the University of Leicester), the father of DNA fingerprinting, found a one in 100 billion chance that there had been a mistake. We would have needed a field of that many sheep. No doubt the National Enquirer would say there are not that many sheep on Earth, therefore Dolly must have come from an alien," quips Alan Colman, research director at PPL Therapeutics Inc. in Roslin, Edinburgh. Cumulina's and Dolly's beginnings differ in several key ways. Dolly's donor nucleus, from a mammary gland, was starved in culture to stall it in G0, a "time out" phase of the cell cycle. Keeping the donor nucleus in G0 was thought to be key to the Roslin Institute's success, but the Hawaii group showed the situation is more complex by using three cell types in this stage--with widely varying results. Nuclei from cumulus cells, which surround an oocyte (a developing egg), supported development to the blastocyst stage (a ball of cells) considerably more often than nuclei from neurons or Sertoli cells (which nourish developing sperm). "There may be something special about that type of cell. Maybe it retains the capacity to stop dividing and then do it again later," suggests Robl. Further experiments used cumulus cells. In the first series of nuclear transfers, the researchers placed 142 embryos in 16 foster mothers, yielding five live and five dead fetuses. A second series transferred 800 embryos to 54 foster mothers, producing 10 healthy pups. The third series was the most visually striking--agouti (coffee-colored) mice donated the nuclei, black mice provided the enucleated oocytes, and the foster mothers were white. As predicted, the cloned mice were agouti, and, of course, all female because the donor nucleus was from ovary tissue. A final series of experiments successfully cloned the clones. Although no one knows just why the experiments worked, Yanagimachi suggests that their microinjection technique and delaying egg activation may have made the difference. Their device "drives the pipette in very quickly, through the zona pellucida (surrounding the oocyte) without deforming it at all. There is no puncture and no damage, to which mice are very sensitive," he explains. "Before we used this approach, 80 percent of the oocytes died at this point. With the injector, more than 90 percent survive," he adds. One to six hours elapsed between nuclear transfer and chemical activation with strontium, which jump-starts development. "They are like sleeping cells that must wake up. During the one to six hours it takes for chromosome condensation, something important happens," says Yanagimachi. That something might be the cell adjusting to the absence of conventional fertilization. Normally, the sperm contributes one set of chromosomes, and the oocyte, on the brink of completing meiosis, has two attached sets of chromosomes, one of which is jettisoned in the form of a polar body when the sperm enters. In cloning, an oocyte that has had its genetic material removed is given a novel, complete double set of chromosomes. Researchers add cytochalasin B to prevent the oocyte from releasing chromosomes, as it would in a polar body. "The whole natural order is broken," explains Davor Solter, director of the Max Planck Institute in Freiburg, Germany, who wrote an editorial to accompany the mouse cloning article (D. Solter, Nature, 394:315-6, July 23, 1998) and had concluded in 1984 that cloning mice was difficult, if not impossible (J. McGrath, D. Solter, Science, 226:1317-9, 1984). "You have to prevent completion of meiosis and keep the chromosomes in, then nudge them gently into a round of DNA synthesis. It is very tricky to manipulate all that," he adds. The panel of agricultural researchers and developmental biologists assembled at the press conference in New York to discuss applications included Yanagimachi, Robl, Colman, and Wall, and professor of genetics and development at Columbia University Virginia Papaioannou. The speakers related how cloning fits into existing biotechnologies. For example, "knocking in" or "knocking out" genes in ES cells takes four steps. This is because the altered ES cell must first be part of a chimeric blastocyst, which develops into a chimeric animal, and if the trait is recessive, crosses must be set up to obtain an animal with two copies of the affected gene in each cell. Cloning would eliminate one step, while greatly increasing efficiency. "With nuclear transfer, you can make all animals in one generation (sheep and cows), and it would take two and a half years to get milk," says Colman, compared to the four and a half to five years it takes now. Cloning research predates even Woody Allen's accurate depiction of cloning a political leader from a nose cell in the 1973 film Sleeper , and plant biologists have been doing it successfully for decades. Many developmental biologists today cite the March 7, 1996 Nature paper from Ian Wilmut's laboratory at the Roslin Institute as a turning point in mammalian cloning. "They used nuclei from sheep embryos, and what was important was that they cultured the nuclei prior to cloning. The popular press ignored this, but it caused quite a stir in the biological community," recalls Virginia Papaioannou , a professor of genetics and development at Columbia University. Adds Ronald McKay , chief of the laboratory of molecular biology at the National Institute of Neurological Disorders and Stroke, of the mouse cloning, "It is not a major new scientific concept, but a great technical achievement." Following are some key papers in the chronology of animal cloning: R. Briggs, T.J. King, "Transplantation of living nuclei from blastula cells into enucleated frog eggs." Proceedings of the National Academy of Sciences, 38:455-63, 1952. J. B. Gurdon, "The developmental capacity of nuclei taken from intestinal epithelial cells of feeding tadpoles." Journal of Embryology and Experimental Morphology, 10:622-40, 1962. J.B. Gurdon et al., "The developmental capacity of nuclei transplanted from keratinized cells of adult frogs." J. Embryol. Exp. Morphol, 34:93-112, 1975. S. M. Willadsen, "A method for culture of micromanipulated sheep embryos and its use to produce monozygotic twins." Nature, 277:298-300, 1979. J. McGrath, D. Solter. "Nuclear transplantation in the mouse embryo by microsurgery and cell fusion." Science, 220:1300-2, 1983. J. McGrath, D. Solter, "Inability of mouse blastomere nuclei transferred to enucleate zygotes to support development in vitro." Science, 226:1317-9, 1984. S. M. Willadsen. "Nuclear transplantation in sheep embryos." Nature, 320:63-5, 1986. K.H.S. Campbell et al., "Sheep cloned by nuclear transfer from a cultured cell line." Nature, 380:64-7, 1996. I. Wilmut et al., "Viable offspring derived from fetal and adult mammalian cells." Nature, 385:810-3, 1997. T. Wakayama et al., "Mice cloned from adult cell nuclei." Nature, 394:369-73, July 23,1998. Cloning applications can include genetic engineering or not. Without genetic manipulation, cloning could be used to assess environmental influences on production traits in livestock by holding genetics constant; transfer elite traits into herds faster than can be done with artificial insemination; and create genetically identical flocks and herds that produce human pharmaceuticals in their milk. And the greater efficiency of mouse cloning--2 to 3 percent compared to the 1 in 277 attempts it took to make Dolly, or the 1 in 500 tries it takes to make a "conventional" transgenic animal--puts the technology in the realm of the economically feasible, if losses occur at the early, in vitro stage, as they do in mice, says Robl. "Novel products" result from tinkering with genes before cloning. For example, removing prion genes can create sheep and cows resistant to scrapie and BSE, making the food supply safer. Pharmaceuticals could also be purer. "We can replace the bovine with the human serum albumin gene, or human antibody genes, so we can get these products from 'immunocows' rather than human plasma," Colman says. PPL Therapeutics is also cloning pigs (from embryonic nuclei, so far) that lack antigens that provoke a human immune response, for use in xenotransplantation. PPL Therapeutics and ProBio America have begun a joint venture that will catalyze commercialization of pharmaceuticals from cloned livestock, with the Scottish arm contributing the expertise in protein production, and the Hawaiian contingent adding the "Honolulu technique" of microinjection. The mouse cloning work opens a new window on early mammalian development. If Papaioannou's responses are any indication, developmental biologists will barely be able to ask questions fast enough. "What happens to a genome as an embryo develops from one cell to millions? How are molecular signals sent and interpreted? How is this reversed? What are the biological and technical limitations to cloning? Can all types of cells be reprogrammed? Must all the genes be reprogrammed? Can some differentiated features be retained and cloning still work?" she asks. Other questions target the end of life. "Are Dolly and Cumulina older than their chronological age? Biological clocks run in association with chromosomes shortening over time. Is Dolly really six years old? She might be a pretty old sheep if her biological clock is from the sheep from which she is derived. Or do we start the process all over again?" asks Wall. Answers may come from the cloned mice, with their shorter-than-sheep life spans. Says Colman of the difficulty in studying aging in mammals, "These animals are not like oak trees, they don't have rings that we can count. With Dolly, the only way we can monitor her age is by looking at her teeth." The press conference speakers stuck doggedly to discussing science and technology, despite persistent pressure by the mass-media press to comment on human cloning. The repeated requests elicited only a terse opinion from Colman that cloning humans is immoral and should be banned. And perhaps because the public is sated on science news in general, and cloning talk in particular, the resulting media reports were low-key compared to the circus that greeted Dolly. The Today Show, for example, considered Monica Lewinsky, deaths of an astronaut and an actor, a heat wave, the latest lottery news, and a film debut to be more important than the cloning of a mouse. At least one bioethicist's reaction was rather tempered too. "There may not be any obvious ethical implications of a cloned mouse. It won't run rampant or have a bad temper. But the long-term implications of this science are enormous," says Glenn McGee, a bioethicist at the University of Pennsylvania. He mentions research into the events immediately following conception, and mice as model organisms, as valuable uses of the technology. But the success in mice adds evidence that human cloning is possible, and Lee Silver, professor of molecular biology at Princeton University, explains why: "It is now clear that cloning is not specific to a single species, and sheep and mice are about as far apart as you can get among placental mammals. Sheep need their embryonic genome by the eight-cell stage, human embryos start using theirs by the four-cell stage, and mice do this by the two-cell stage. There was worry that if reprogramming DNA took a long time, mice and humans would be unable to use their embryonic genomes. So there is now no longer a reason to expect that humans couldn't be cloned as well." The idea of taking a person's cell, growing an embryo, and coaxing ES cells to yield replacement parts--just one possible cloning scenario--is disturbing. But the hope among researchers is that cloning will lead to an understanding of early development that will ultimately make it possible to bypass whole organisms, especially since adult tissue harbors stem cells too. Concludes Colman: "My fantasy vision is to investigate if it is possible to change one adult cell into another without the route through the embryo. It is a fantasy--but so was Yanagimachi's work just two years ago." Ricki Lewis, a freelance science writer based in Scotia, N.Y., is the author of several biology textbooks. She can be reached online at rickilewis@nasw.org.

Mammalian Cloning Milestone: Mice from Mice from Mice

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