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AUTHOR: RICKI LEWIS, pp.18 In just over a decade, genetically engineered mice have brought dramatic changes to the biomedical sciences, offering basic researchers ways to detect the influences of single genes and more clinically oriented investigators compellingly close models of human disease. The following suppliers are among those providing transgenic and knockout mice to researchers. Charles River Laboratories 251 Ballardvale St. Wilmington, Mass. 01887 (508) 658-6000 Products: OncoMice (five strains, transgenes are various oncogenes); apo-A-1 (transgene is human apolipoprotein A-1); ImmortoMouse (transgene is an SV40 gene allowing immortality of cells in culture). Call for details and prices. DNX 303B College Rd. East Princeton Forrestal Ctr. Princeton, N.J. 08540 (609) 520-0300 Fax: (609) 520-9864 Products: Premium mouse service (microinjection) with PCR $7,250; with slot blot analysis $7,500; with southern blot analysis $8,000. Shipping and handling $100. Basic mouse service $4,000 (researcher identifies transgenics). Exemplar Corp. 1 Innovation Dr. Worcester, Mass. 01605 (508) 755-0550 Products: Transgenic mouse models in development for oncogenes, Alzheimer's disease, AIDS, and genetic toxicology. Call for information on expected availability. GenPharm International 297 North Bernardo Ave. Mountain View, Calif. 94043 (415) 964-7024 Fax: (415) 964-3537 Products: Strains include p53, pim-1 oncogene, immunodeficient strains. $100-200 per mouse, plus $1,000 Research Breeding Agreement. The Jackson Laboratory 600 Main St. Bar Harbor, Maine 04609 (207) 288-3371 Products: Transgenic and knockout mice are $65-$75 per breeding pair. "These mice are, quite simply, extraordinary," says Joseph Perpich, vice president for grants and special programs at the Howard Hughes Medical Institute (HHMI) in Chevy Chase, Md., many of whose investigators use the new mice. Unlike the laboratory mice that have long been a staple of biology, genetically engineered mice carry genes specifically selected and imported into their genomes through a complex process involving DNA delivery, embryo manipulation, and classic genetics. And the technological challenges and labor necessary to create each line of mice have led to a problem--how to supply these custom-designed animals to the growing numbers of researchers requesting them, while keeping costs down. When companies began licensing rights to market these mice and then charging scientists stiff fees and imposing difficult restrictions on their use, researchers protested (C. Anderson, Science, 260:23, 1993; J. Travis, Science, 256:1393, 1992). Now, however, a new program at the Jackson Laboratory in Bar Harbor, Maine, as well as a breeding center there backed by the National Institutes of Health, should increase the availability and affordability of genetically engineered mice. In addition, researchers can choose among several commercial vendors or develop their own mice. There are two kinds of new laboratory mouse models: Transgenic mice bear a foreign gene inserted randomly into their genome; so- called knockout animals carry an inactivated form of a gene that replaces a functional version. Both technologies begin with identifying and isolating a gene of interest. The gene is then delivered, usually by microinjection, into a target cell. In transgenic technology, the target cell, a fertilized ovum, is implanted into a foster mother. The investigator then selects and breeds to each other the heterozygous offspring--those with one copy of the inserted, recessive gene, the transgene--to produce homozygotes that express the recessive trait the gene encodes. Transgenic organisms often are used for what has been called "pharming"--producing a pharmaceutical substance such as human clotting factor in their milk, for example. They also serve as models of human disease. Perhaps the best-known transgenic is the OncoMouse, also known as the Harvard Mouse, developed by Philip Leder at Harvard Medical School in 1988 (E. Sinn, et al., Cell, 49:465, 1987). The breast-cancer-prone rodent was licensed to E.I. Du Pont de Nemours and Co. Inc., Wilmington, Del., and distributed by Charles River Laboratories, Wilmington, Mass. Along with its innovative genetic programming, the mouse was delivered to researchers with problematic restrictions, including a so-called reach-through clause claiming royalties for Du Pont on any products or inventions using the mice. Gene targeting, to create the knockout mice, is a bit trickier than transgenic technology. It relies on a natural process called homologous recombination, in which a DNA sequence seeks its complement on a chromosome, and then switches places with it. The targets are mouse embryonic stem cells, taken from four-day-old mouse embryos. At this stage, the cells will take up foreign DNA, and they can then be introduced into another embryo to continue developing as part of it, still capable of differentiating into any tissue (M.J. Evans, M.H. Kaufman, Nature, 292:154, 1981; G. Martin, Proceedings of the National Academy of Sciences, 78:7634, 1981). To engineer a knockout mouse, a researcher transfers an inactivated gene into an embryonic stem cell, cultures that cell, then adds single cells to embryos from mice of a different color. The hybrid embryos complete development in a foster mother. Progeny containing some cells with the targeted gene are easy to spot--after birth, they have splotches of the color of the manipulated mouse in the background color of the recipient embryo. The bi-colored mice are next bred to albino mice. Completely pigmented progeny have the targeted gene in each cell--but only one copy. These pigmented mice must then be bred to each other. According to Mendel's laws, each offspring has a one-in-four chance of being a sought-after homozygote--a mouse with two copies of the targeted gene. Knockout mice model the most severe forms of human inherited disorders because they express a complete lack of gene function (J.N. Snouwaert, et al., Science, 257:1083, 1992). Particularly intriguing are knockout mice that bear inactivated genes thought to be essential for life--and yet survive (K.L. Philpott, et al., Science, 256:1448, 1992). This was the case for the p53 knockout mice developed in the laboratory of Allan Bradley at the Baylor College of Medicine in Houston (L.A. Donehower, et al., Nature, 356:215, 1992). Lack of p53, a tumor suppressor gene, can cause a variety of cancers in humans. So, researchers were surprised that mice with a double dose of knocked-out p53 appear quite healthy. The mice do develop malignancies as they age--but not more often than do humans. Now, the possibility that the mice might possess some undiscovered genetic factor able to compensate for lack of p53 function is stimulating a new set of research questions. The p53 knockout mouse was the cause of the recent controversy over mouse pricing. Bradley's lab, unable to meet requests from colleagues for the p53 knockouts, licensed the mouse to GenPharm International in Mountain View, Calif. Because of high licensing fees and the considerable cost of maintaining the animals, GenPharm's p53 knockouts sold, in 1992, for $100 per mouse. And at first, researchers were not permitted to breed the mice other than to produce a few offspring to test the mice for birth defects. Anger over GenPharm's pricing and restrictions coalesced at the Mouse Molecular Genetics meeting at Cold Spring Harbor Laboratory, Long Island, N.Y., in August 1992, in an impromptu session attended by more than 300 researchers and chaired by NIH director-designate and Nobel Prize-winning University of California, San Francisco, geneticist Harold Varmus. "The Cold Spring Harbor meeting was critical," says HHMI's Perpich. "There had been ad hoc meetings of scientists all over the country [to protest GenPharm's policies], but nothing had been crystallizing." One alternative to GenPharm brought up at the meeting was to involve the Jackson Laboratory, a nonprofit facility founded in 1929 to supply inbred strains of "Jax" mice for research, in producing supplies of genetically engineered mice. "At Cold Spring Harbor, Harold Varmus spoke to Howard Hughes Medical Institute's president and vice president about making a [financial] contribution" to the Jackson Laboratory to develop the mice, Perpich says. "It would be important for our own investigators and for the broader scientific community." With HHMI willing to provide funding to set up a program to supply the mice and researchers becoming more vocal in their objections to mouse prices, by the spring of this year GenPharm was already bowing to the pressure. Company president David Winter announced that, for a $1,000 annual fee, researchers could freely breed their GenPharm mice. "We no longer limit the number of offspring a researcher can breed, and [scientists] don't have to disclose the nature of the work. We let researchers do what they want with the animals," says Anthony P. Cruz, product manager of the transgenic laboratory at GenPharm, pointing out, too, that the company never had a reach-through clause similar to Du Pont's. Meanwhile, HHMI was rushing through a $1.2 million grant to the Jackson Laboratory to implement a mouse-supply program. "By May, it was all set, and the check, in full, was sent out three weeks later," says Perpich. Unexpectedly, support for the Jackson program from other sources took off. Organizations funding research into specific human disorders, from orphan genetic diseases to major killers, began to recognize the power of these mice as disease models. Dollars poured in, first from the March of Dimes, and soon after from the American Cancer Society, the American Heart Association, the Multiple Sclerosis Society, the Cystic Fibrosis Foundation, and others. The Maine facility got to work immediately. "The Jackson lab is making a concerted effort to start acquiring these mice, maintain them, and make them available," says Mario R. Capecchi, the HHMI investigator and professor of human genetics at the University of Utah, Salt Lake City, who pioneered gene targeting (M.R. Capecchi, Science, 244:1288, 1989; S.L. Mansour, K.R. Thomas, M.R. Capecchi, Nature, 336:348, 1988). "An important function will be to cryopreserve embryos, so that breeding need not be repeated over and over," he adds. "Some mice are available now," says John Sharp, superintendent of the induced mutant resource at the Jackson lab. "A researcher would order them like any Jax mice. We are trying to base prices on the amount of genetic typing that has to be done. Most range from $65 to $75 for a breeding pair of heterozygotes. And, as with all our mice, the researcher can keep the progeny." The "Jaxp53" strain, for example, knocked out for the p53 gene, sells for $60 per breeding pair. It was developed in the laboratory of cancer geneticist Tyler Jacks at the Massachusetts Institute of Technology. Soon there will be even more support for genetically engineered mice. NIH's National Center for Research Resources awarded a grant to the Jackson Laboratory earlier this fall to create a National Resource for Transgenic Animals. This program will provide transgenic and knockout mice at cost to basic researchers. Researchers seeking genetically altered mice have alternatives to commercial suppliers and the Jackson Laboratory. DNA Biotherapeutics Inc. in Princeton, N.J., offers contract services in developing transgenic mice. "Injecting DNA, screening the progeny, and identifying progeny costs $7,250 to $8,000," says Mark E. Swanson, associate scientific director for transgenic animal development. "But we find that most academic labs can't afford this. So we offer, for $4,000, to inject the gene and send progeny of injected embryos, and the researchers screen to identify the transgenics." DNX, also based in Princeton, offers yet another option--a $2,000 program underwritten by the National Institute of Child Health and Human Development (NICHD). Researchers with proposals accepted by NICHD may send DNA to the NICHD Transgenic Mouse Development Facility operated by DNX. Some companies developing strains of transgenic or knockout mice have entered into collaborations with research institutions as they move toward making the mice commercially available. This is the case at Exemplar Corp., Worcester, Mass. Exemplar's proprietary transgenic mice currently are being used in research on AIDS, cancer, and Alzheimer's disease, although they are not yet available for purchase. "We have given them to institutions with whom we've entered into a relationship," says Paul Leibowitz, Exemplar's senior vice president of research and development. "They can't give them to a third party, but they are free to publish on anything." For those laboratories with the combination of expertise and technology necessary to engineer their own mice, that too will get easier, Capecchi says, as people become more adept at the manipulations of creating transgenics and knockouts. "And the price will continue to go down as people become better at it," he adds. The diverse suppliers of genetically engineered mice are likely to complement each other rather than compete, experts in and users of the technologies agree. Sharp of the Jackson Laboratory predicts a stratification of the market, with commercial vendors focusing on mice needed for research on such prevalent disorders as cancer and heart disease, and subsidized sources pursuing the rarer genetic conditions. Ricki Lewis is a freelance science writer based in Scotia, N.Y.

Knockout Mice Adding New Punch To Genetic Research

Nov 14, 1993

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