See How They Run

Date: November 23, 1998Tools Breeders of transgenic mice assist researchers by isolating genotypes of significant interest and maintaining a ready supply of laboratory animals. Mountains will go into labour, and a silly little mouse will be born. -Horace, Roman poet Rodents have been the bane of human civilization from its inception. From the infestations of the great Roman granaries at Alexandria to the Mongol trading routes that brought the Himalayan brown rat and its constant companion the Black Plague into Europe, mouse and rat populations have flourished wherever humankind has taken root. Throughout history, where people went, rodents were preparing a place for them, or vice versa. Living on seed grain, devouring cattle and chicken feed, and nestling in bedding and linen, mice have been master scavengers. For once, human beings have turned the tables on these diminutive pests, making their appetite for reproduction a positive benefit to society. These small creatures are being transformed into virtual bioreactors for investigating the function of drugs in living organisms. Before you reach for your Limburger cheese, however, the common field mouse or swamp rat is insufficient for such purposes. The wildly erratic mating behavior and filthy living conditions of wild rodents make harvesting them impractical for research. Fortunately, members of a select group of companies have committed themselves to breeding very pure strains of transgenic mice for basic research. By selecting out specific phenotypic as well as genetic characteristics, scientists have been able to produce elegant nuances of behavior and complexion. Bred and monitored under the strictest conditions, transgenic mice join their cousins, inbred strains of mice, to serve as templates for fundamental research in genetics. How does one go from a garden-variety pest to a space-age transgenic mouse? There are two ways. The first method involves selecting a piece of DNA that expresses a particular gene of interest and transfecting that DNA into embryonic stem (ES) cells. The ES cells are then injected into a blastocyst, which, in turn, is implanted in a foster mother. The resulting progeny are chimeric for the desired characteristic, and are inbred to produce a pure homozygous stock of animals. The alternative method, which is more commonly used, is identical in all respects except that instead of a blastocyst, a fertilized egg microinjected with pronuclei gene-carrying vectors) is implanted in the mouse. Researchers investigating a host of problems can find transgenic mice in an abundance of shapes, forms, and dysfunctions. There are diabetic mice for studying pancreatitis, nude mice for looking at immune disorders, and even hypertensive mice for investigating heart disease. But which mouse, you might be asking yourself, is the right mouse for me? In the following profile, some of the leading breeders of transgenic mice will be discussed with regard to their products. From the earliest stages of build-your-own mouse technology, Taconic has been providing transgenic mice for the research community. In 1988, the company was the first breeder to have a successful large-scale production of transgenic mice with human oncogenes. In 1995, Taconic became the first fully licensed commercial producer of transgenic and knockout mice, and in 1996, the Taconic Transgenic Exchange allowed researchers to create and share transgenic and knockout rats and mice. Taconic offers a growing list of knockout and transgenic rats and mice including p53, rag-2 and TNF-ice® TNF-® mice, along with other transgenic strains that are distributed for and in association with Chrysalis DNX®. All models are available with fully defined health and genetic profiles, as well as hassle-free label licenses. The Taconic Emerging Models program involves the development, characterization, and distribution of newly developed transgenic rats and mice. Specialized breeding services are also open to investigators for their transgenic models. Taconic offers a comprehensive range of breeding, genotyping, and model development services. For many researchers who were taught that in order to be a good scientist you have to get your hands dirty, the colony maintenance and development services at Charles River Laboratories (CRL) might seem a little bit like cheating. What some scientists find hard to accept is that the Diane Fossey scenario of going off to the jungle to live with the apes is not necessary. In fact, one can do viable research without being immersed in the "blood and guts" duties of maintaining animal populations. Molecular biologists who have essentially received their postdoctoral training in animal husbandry and, in addition, are board certified rat surgeons may have some difficulty adjusting to the new method. CRL provides breeding strategies, colony maintenance, production breeding, backcrossing, and intercrossing of transgenic strains for an ever-growing number of scientists who are tired of being mother, zookeeper, surgeon, and matchmaker all rolled into one. The company also offers genetic monitoring that characterizes the genetic profiles of rodent colonies, as well as embryo cryopreservation in case the rodent colony is compromised by disease or genetic changes. CRL has even begun implanting subcutaneous transponders in its mice so that each individual can be "unequivocally identified." Many researchers who haven't heard about the range of CRL's colony maintenance services will be more familiar with its rederivation method. Pioneered by CRL's founder, Dr. Henry L. Foster, the technique that removes parasites from rodent colonies is used in conjunction with a health monitoring and animal quarantine program to ensure that customers are getting the purest animals possible. Lexicon Genetics burst onto the scene in 1995 with the objective of capturing a piece of the market in transgenic mice. In short order, it launched a homologous recombination program resulting in collaborations with over 25 clients to create knockout mice. One of Lexicon's most ambitious projects is its plan to use gene-trapping technology to produce 500,000 mutant mouse embryonic stem cell clones. When cataloged in its Omnibank® database of gene function, the combination of these potent forces will be a powerful tool in the science of drug discovery. Presently, Omnibank® contains over 10,000 clusters, which represent approximately 10 percent of the mouse genome. In addition to its formidable complement of mouse resources, Lexicon provides products to help researchers start building their own transgenic lines. The Mouse Kit® allows scientists to duplicate the techniques and procedures using the same stem cells, feeder cells, and buffers that Lexicon uses in its own transgenic research. The company has also packaged gene targeting plasmids in the pKO Scrambler Series® that make the generation of targeting vectors faster and easier. These products are distributed by Stratagene and Takara Shuzo Co., Ltd. Whether you're looking to delegate research activities or start your own colony, Lexicon has options for you. Chrysalis -- DNX Transgenic Sciences patented the process of pronuclear microinjection that is now the most widely used method of producing transgenic mice (Gordon, J.W. et al., Proc. Natl. Acad. Sci, 77:7380-7384, 1980; Palmiter, R.D. et al., Nature, 300:611-615, 1982). This innovation made high-throughput production of transgenics a practical reality, expanding the breadth and scope of what was originally a niche market. By targeting particular genes to see if they have a function relevant to human health concerns, Chrysalis has made a number of important discoveries. "What we really focus on is to screen potential therapeutic target genes," said David Grass, associate director of transgenic science at Chrysalis. "Mice don't have CETP [cholesteryl ester transfer protein] endogenously. Adding the gene for CETP lowers HDLs and raises LDLs." The DNX Transgenic models ApoB and CETP/ApoB, which express human-like serum HDL/LDL distribution, have led to findings about foam cell accumulation, cholesterol crystals, and fibrotic lesions. Although some argue that mouse pathology cannot be directly correlated with human pathology, the similarities between the two are striking and, at the very least, offer some clues to the mechanisms of genetic disease. Chrysalis -- DNX Transgenic Sciences makes several hundred transgenic lines each year--some for its own research activities, some for its custom model programs, and the bulk for its production program (from genes sent to the company by researchers throughout the world). In addition, it has customized gene-targeting capabilities for over 20 pharmaceutical and biotechnology clients. Chrysalis provides complete molecular biology, breeding, and characterization for its transgenic projects. Chrysalis recently scaled up its transgenic production capabilities to be able to screen hundreds of potential therapeutic target genes per year for their involvement in human disease conditions. Chrysalis is focusing on establishing research partners to identify and validate new therapeutic targets for drug discovery efforts. Genome Systems is a provider of mouse cloning products that includes everything from well plates and high-density filters to the latest in cDNA libraries. Its contract transgenic programs have complete gene knockout or knockin services from project design through gene cloning and characterization, targeting vector construction, ES cell targeting, inbreeding, and chimera production. This is a service for researchers who want to maintain their own colony of animals but need the expertise of a proven breeder to do the initial work of deriving and testing a transgenic strain. Genome Systems attributes much of its success to the ability of its ES cells to go germline after blastocyst injection or morula aggregation. Not only does GSI have 129/SvJ ES lines that have proven effective in generating targeted mutations, but they also have a stable C57BL/6 derived ES line that is the preferred strain for immunological study. Robin Wesselschmidt, product manager for Genome Systems, emphasized the flexibility of her company's services. "We do a custom service; we make the mouse that our customers want," she said. Jackson Laboratory boasts the largest freezing bank for transgenic mice in the world. With over 400 strains of transgenic and knockout mice designated for cancer research, immunology, cardiovascular disease, embryology, toxicology, neurobiology, and metabolic diseases, Jackson Laboratory affords a wide range of possibilities for the research community. Jackson Laboratory's Induced Mutant Resource (IMR) receives mice from all over the world. These are added to the lab's vast repository of transgenic mice until it is decided that the specific characteristics of the mouse make it a scientifically relevant candidate for research. Once this decision is made, the mouse is listed on Jackson Laboratory's home page; the company begins taking orders and starts the process of rederiving and building a colony. Although Jackson Laboratory does not create its own transgenic mice, its researchers are open to suggestions for acquiring new mutants. If they don't have the strain in their catalog already, they will make an effort to obtain mice for which there is a sudden demand. At present, Jackson Laboratory increases its selection by eight new strains every month. The immediate future of transgenics seems to involve the use of a technology called Cre-lox. The excitement surrounding Dupont's discovery is justified by its ability to transfect postmitotic cells. The recombinase system that is able to remove or insert DNA anywhere in the genome is already being used by Jackson Laboratory and Lexicon to make gene-specific knockout mice (E. Russo, The Scientist, 12[18]:1, Sept. 14, 1998). The problem with previous methods is that the removal or addition of a specific gene at the embryonic stage meant that all of the descendants of the original totipotent stem cells had the same altered gene characteristics in the mature animal. The universal expression of a mutant transgene is not always desirable. In addition, many genes of interest play a critical role in the development of the embryo. Until now, these genes have not been investigated because their substitution or deletion has resulted in the death of the animal. What makes Cre-lox so significant is that it can be used at a later stage in a mouse's development, when specific tissues can be targeted instead of the entire organism all at once (M. Barinaga, Science, 265:15, 1994). By this time next year, the annual review of transgenics may tell a completely different story. With Cre-lox technology beckoning researchers to explore new methods and procedures, 1999 may well be remembered as the year of the mouse. Tools Author Brent Johnson can be reached at bjohnson@the-scientist.com

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