Issues Posed by the New Mouse Genetics, and Possible Solutions
The development and proliferating use of genetically altered mice have raised a number of important issues and concerns that affect the ability of researchers to share this increasingly important resource. Three key subjects of concern are intellectual-property rights, safe and efficient distribution of the mice to researchers, and the handling of the proliferation of strains.
The workshop aired issues and explored problems in each. Participants did not try to resolve the problems identified, but they did identify and discuss ideas for solutions. The ideas discussed in this chapter arose at the conference and might be useful to those who will be charged to answer the questions posed.
The Harvard Oncomouse and Other Mouse Patents
Harvard University was granted a patent on the so-called oncomouse in April 1988. It was the first animal ever given patent protection by any country. The mouse has been transgenically engineered to be especially susceptible to cancer. The advent of the patent for the Harvard mouse opened the door to the patenting of animals both here and in Europe. The considerable, continuing, and widespread controversy attendant on that practice is centered largely on ethical and economic concerns. Within the scientific community, a major focus of concern about the granting of patent protection of animals has been scientific access to and exchange of research materials.
The principal inventor of the oncomouse is Philip Leder, who was appointed to the faculty of Harvard University Medical School in 1981. In conjunction with his appointment, DuPont had given Harvard $6 million for the support of Leder's research. A Harvard spokesperson stressed that the grant was “sympathetic with academic freedom in the pursuit of knowledge” and that DuPont would impose no restraints on the recipient's freedom to talk about or publish his work. Harvard had earlier suffered a negative reaction when it tried to commercialize the products of another Harvard biologist's research. The arrangement with DuPont was a retreat to more conventional practices like the one that Harvard had entered into with Monsanto in the mid-
1970s. In this arrangement with DuPont, Harvard would own any patents that might arise from Leder's research, but DuPont would be entitled to exclusive license on any and all such properties.
During the next 2 years, exploiting the transgenic technology developed by Gordon and Ruddle, Leder and his collaborators developed the oncomouse by inserting the MYC oncogene attached to a specific promoter into the embryo of a normal mouse. The promoter is a mouse mammary-tumor virus-promoter that is expressed directly in breast epithelial cells, and mice with this oncogene reproducibly develop breast cancer. Leder 's work was not aimed at devising a patentable product; it derived from research on Burkitt's lymphoma that he had begun at the National Institutes of Health. However, once his research was successful, Leder recognized that it might have commercial possibility.
Near the end of 1983, Leder brought his mice to the attention of the Office of Technology Licensing and Industry-Sponsored Research at the Harvard Medical School, which had recently been created in line with Harvard's thrust to commercialize the results of research done in its laboratories. The office assembled a small group, including Leder, several DuPont intellectual-property lawyers, and Paul Clark, a patent attorney who was Harvard's principal outside patent counsel. Clark believed that it was important to patent the mice to give Harvard and DuPont, which was the licensee, all the legal rights to which they were entitled. He further believed that claims on methods of using the mice or on plasmids, although of some importance, would not have protected the invention adequately by themselves. The reasoning among patent lawyers is that it is best to protect the product as well as the processes used to create it, to prevent competitors who are using different processes from trying to develop similar but competitive products.
Clark also saw that Leder's transgenic animals were new compositions of matter made by humans –like the bacteria on which the Supreme Court had ruled in 1980. Genes are matter; they are DNA. The oncomouse does not occur naturally; it was made by humans. It was therefore a new composition of matter because a human being had inserted a new chemical into the genome of the mouse. Consequently, it could be patented.
On June 22, 1984, on behalf of Harvard, Clark filed an application for a patent on Leder's invention. The main utilities that he claimed were straightforward but very broad. They included the use of such animals as sources of malignant or protomalignant tissue for cell culture and as living systems on which to test chemicals for carcinogenicity or, in the case of substances such as vitamin E, for the power to prevent cancers.
Clark had not been conservative in what he claimed as the actual invention. It was not simply a transgenic mouse with an activated MYC gene; it was any transgenic mammal, excluding human beings, that contain in all its cells an activated oncogene that had been introduced into it or an ancestor at
an embryonic stage and the patent that was issued to Harvard in 1988 was that broad in scope.
At the outset, DuPont made the oncomouse available for basic research for a comparatively low fee–about $50–with no restrictions. When Charles River Laboratories began talking to DuPont in 1988 about supplying the oncomouse, all the ingredients for success seemed to be present: the involvement of Harvard and DuPont, the first patent of an animal, a commodity labeled the “product of the year” by a major financial magazine, and the opportunity to distribute the animals worldwide.
At the beginning, the oncomouse was a small part of the DuPont plan for a large biotechnology group that would sell reagents and develop other mouse models. DuPont instituted a practice of requiring royalties on products derived from use of the mice. As time went on, DuPont, which had planned to use only Charles River's breeding and distribution capabilities, turned the mouse over to Charles River for development but retained the rights to downstream royalties (those defined on the basis of sales or profits of products developed with the mouse as a research tool). That occurred as DuPont dissolved its biotechnology group.
Charles River scaled up to produce five different lines of oncomice –three breast-tumor models, a B-cell lymphoma model and a papilloma model. The restrictions have become so limiting on downstream revenues from new products that there are few purchasers of the oncomice. One possible reason is that some scientists may ignore the patents and create mice as they need them. However, the potential for revenues is very limited.
The Pros and Cons of Patenting and Licensing Mice
“If universities are in the business to try to make a lot of money and replace their research budgets with royalties, they are in the wrong business. There are easier ways to make money.”–Lita Nelsen
In the wake of the Bayh-Dole Act, some academic institutions deepened relationships between their biology departments and industry. Some faculty members formed research and development companies. And universities renewed their pre-World War II interest in generating parts of their research budgets from income on patented products of their laboratories.
Even before the patenting by Harvard of the oncomouse, some investigators had envisioned that a mouse from their laboratories might be a major producer of revenue. However, large profits from mouse patents have not yet materialized, and some wonder whether they ever will.
Universities are making, on the average, less than 1% of their research budgets from royalties on patented inventions from their laboratories. Most universities are breaking even, and some are losing money on their com-
mercialization efforts. Some universities with successful products raise about 2-3% of their research budgets from income from these products.
Overall, the prospects of developing a commercially successful mouse are slim. The expectations of academic developers are often unrealistically high. A successful mouse might make tens of thousands of dollars but not the millions that academic developers might believe that the mice are worth.
For genetically altered mice to be of widespread use in such applications as drug-testing or development of new therapies, validation is important, that is, proving that the genetic trait that has been introduced is reproducibly expressed and that the mouse is a good model for the human disease that it was designed to study.
One potential barrier to commercial use is the existence of well-established alternatives. Nontransgenic mice are now used in studies regulated by the Food and Drug Administration. Because they are established and accepted for this use, it might be difficult to displace them with transgenic mice. Thus, another path to a potential market would be blocked.
Another potential barrier is that a transgenic mouse often becomes technologically obsolete in 1-2 years and is replaced by another model. Obsolescence can occur easily if, while one company is expending resources to develop a strain for sale, another produces a mouse or even a rat on a better background strain or one that has a slightly better gene construct or integration-expression site. Obsolescence might occur before income from the mouse has returned the costs of development.
The marketability of transgenic mice is affected by the practices of investigators. Academic scientists who want to use transgenic mice might develop their own. They might not feel restricted by existing patents or believe that they will be challenged on their activities because they use the mice for research and not for sale. Not only are the investigators not customers for such mice, but many will freely share mice with their colleagues. In one instance, an investigator distributed a potentially valuable model to colleagues with the proviso that they would freely pass the mice on to still other colleagues. That saved the original investigator much time and money that would have been required for breeding and shipping.
There is a small market for genetically altered mice in the experience of the Jackson Laboratory. Sales number 1,000 mice a year for only a few mutants, including the models of diabetes, obesity, and lymphoproliferative disease. The Mouse Mutant Resource at the Jackson Laboratory, which has 325 mutants, sells an average of 31 mice per mutation per year. The sale of such mice clearly is undertaken not for profit, but as a service to the research community.
Overall, the Jackson Laboratory distributes about 500 different mutant animals and strains in a given year. Most do not yield a profit, but their distribution is made possible by large modestly profitable sales of enough of
the most widely used inbred strains, such as C57 BL/6, to support their distribution.
An example with sales between dozens and many thousands of mice comes from the Fox Chase Cancer Center. The center announced the introduction of human cells into the SCID (severe combined immuno-deficiency) mouse in 1988. It now licenses the mouse worldwide through all the major mouse-breeding companies. Starting in 1983, the center distributed the mouse itself through material transfer agreements. After 1988, the large number of requests for the mouse led the center to license it nonexclusively and, at first, with pass-through rights. Using bailment mechanisms, whose purpose is to transfer possession but not title to the mouse, Fox Chase distributed some 20,000 in 1992. Fox Chase wished to recover the development costs and the costs of maintaining the mouse for 5 years. Therefore, the center no longer attempts to retain rights to future applications of the mouse.
Given the picture of unpromising profits and a small market for transgenic mice that are essentially resources for a research laboratory, one participant mused, “Why would anybody ever go to the trouble and expense of making any of these mice available to anybody?” But with government encouragement of commercialization through Bayh-Dole and other means, a Patent Office that looks favorably on applications for patents of living things, and the increasing need to seek research funds from new sources, investigators and their universities might continue to harbor expectations that their mouse is a “million-dollar ” bonanza.
Many scientists feel antipathy toward the patenting and licensing of laboratory resources, and both scientists and lawyers suspect that patenting these resources reaches too far into the research phase of research and development–that it usually makes better sense to wait until a product is closer to being marketed before taking out patents. Nevertheless, present law essentially impels patenting. Furthermore, for the companies that are inextricably linked to academic institutions in the biotechnology revolution, patents and licensing arrangements are a valid and proven tradition, just as the free, early sharing of resources and knowledge is for academic scientists. How much of a burden does the need to obtain patent licenses impose on the research community, and how much of a burden can the community learn to live with?
The role of private industry in the development and distribution of genetically altered mice for use in research is a critical issue in need of discussion. Is private investment necessary or desirable either to ensure that new strains of mice are developed or to reproduce existing strains and deliver
Box 4.1. Possible specific actions to address licensing issues in basic research:
them to researchers, or do patentholders or their licensees merely interpose themselves as toll collectors in a system that would work adequately or better without them? Some say there is an important role for private firms to play in this context. The distribution of genetically altered mice is not as simple a matter as distributing cell lines or yeast strains, which investigators can do on their own with minimal interference with their research. If the involvement of private firms is needed, do private firms that develop or distribute genetically altered mice need patent rights to make these activities profitable? Or, more narrowly, do they need to enforce their patent rights against researchers to make a profit? Do they need to enforce their patent rights against researchers in government and university laboratories or against researchers who are working under government grants? Can they recover an adequate profit by exploiting their patent monopolies in other markets?
A number of possible specific actions were discussed (see Box 4.1).
DISTRIBUTION OF GENETICALLY ALTERED MICE
Problems and Expenses
“Mutants and specialized genetic strains of mice for research have been around for many, many decades. The problems of getting them from one investigator to another are not novel. What is novel is the flood of new mutants and how we are going to handle them.” –Ken Paigen
Investigators continue to share mice despite patent restrictions, mice are available if one can pay the price (and can be made anew in the laboratory if not), exciting discoveries continue to flow from laboratories, and some money is being made by some universities, individuals, and firms. Companies are able to obtain models for their research and the development of pharmaceuticals, and several companies are working on the development of “bioreactor” transgenic animals for the production of useful proteins.
Still, real concerns were identified in the workshop. How, in light of the relative ease of producing a multitude of genetically unique laboratory animals, can we manage the abundance? How do we name them, store information about them, distribute them under whatever conditions are required, maintain stocks, decide which to keep and which to dispose of, ensure that distributed strains are free of disease, and validate that a given strain is of a particular genetic makeup? Should there be some central repository? How is all this to be paid for? What are appropriate terms and restrictions on those who receive these animals?
A number of needs and problems are common to any distribution mechanism, whether it be via individual investigators, nonprofit or government laboratories, or commercial firms. The key issues include genetic charact-
erization, reproductive performance, variation in demand, control of disease, shipping, and meeting regulations. Addressing these issues might require 6 months to a year of work, with attendant costs, before a mouse strain is ready for distribution.
Because of their unique characteristics and requirements, genetically altered mice are more expensive to produce than normal strains. Economy-of-scale problems are associated especially with the production of rare strains. For such distributors as the Jackson Laboratory, the sale of large numbers of some strains of mice is essential to help to support the distribution of many others.
Before any transgenic strain can be usefully distributed to researchers, it must be genetically characterized and validated; this can be both complex and expensive. Transgenic lineages can rarely be distinguished by traditional genetic methods, such as biochemical monitoring, but can be identified by DNA analysis. Transgenic lines will have different loci of integration and different copy numbers, which identify the strain. Targeted-mutation lines will show DNA polymorphisms characteristic of the mutation.
It is necessary to maintain an identification both of lineage and of individual animals by genomic DNA analysis based on Southern blots and the polymerase chain reaction. With transgenic animals, it is sometimes possible to generate viable homozygous mice so that DNA analysis need be performed only on a random selection of animals every generation to verify the stability of the presence of the gene in question.
Keeping meticulous pedigree records is necessary, but it is not sufficient to ensure that a strain will express the transgene. Expression will vary dramatically even among siblings and can be affected by many factors, such as genetic imprinting, the kind of background strain, aging, and hormonal activity. Expression analysis can be very challenging. Using a bioassay or visible expression is the only way to be absolutely sure that the gene is expressed.
With targeted mutations, homozygosity often causes lethality; this possibility makes it necessary to breed from heterozygotes and perform DNA analysis of offspring.
Reproductive performance is often a problem with transgenic and mutant mice. Problems can result directly from the integration of a foreign DNA sequence or from the expression of the gene. Many mice need assistance in reproducing through such mechanisms as in vitro fertilization and embryo transfer, hormone administration, gonadotropin administration, or the use of foster mothers. In the future, one method for handling these problems will be to use binary breeding models, in which neither parental strain expresses the gene, but the offspring of a cross between them does.
Another common need of any distributor is to meet cost-effectively the inevitable fluctuations in demand for a strain. For example, with the
announcement in 1988 of the implantation of human cells in the SCID mouse, Fox Chase was overwhelmed with requests and could not meet demand.
Maintaining a large breeding colony of a specific genetic alteration to meet occasional demand is not cost-effective. Furthermore, strains can become obsolete quickly as new models are developed, and there can be a loss of desired expression as mice are bred across generations. In many cases, cryopreservation of embryos or gametes is likely to be much more cost-effective than maintenance of breeding colonies. Present methods of mouse-embryo cryopreservation can yield in vitro survivals of over 90% and, depending on the fecundity of the mouse strain itself, 60% or more of cryopreserved embryos transferred into recipients develop into live young. Nevertheless, additional research is needed to improve the cryopreservation of mouse sperm and oocytes. Such research might reduce the costs of maintaining large stocks of mice dramatically and make it feasible to preserve almost every mouse that is produced.
Control of pathogens and disease is a critical issue for potential distributors. Animals with novel phenotypes might carry diseases or pathogens that mice usually do not carry, and a disease might affect research results without being obvious. Many industrial and pharmaceutical companies will not accept animals that are not certified to be free of specific pathogens.
Proper barriers in a colony are essential to prevent both the spread of pathogens and accidental interbreeding. Barrier systems can involve such features as special filtering mechanisms; sterilization of food, water, and bedding by autoclaving or radiation; use of sterilized clothing by workers; and even keeping workers from owning particular kinds of pets.
Other issues common to any distribution system are the protection and care of the mice or embryos during shipment–food, water, bedding, and temperature control must all be considered–and the meeting of regulations. Great Britain, for example, has strict regulations designed to prevent the introduction of rabies to the island.
Possible Solutions: Advantages and Disadvantages of Different Distribution Options
Those who make the mice available include the individual investigators who share the mice that they have developed; companies that have traditionally marketed specialty mice, such as Charles River Laboratories; biotechnology companies that may see transgenic mice intended for laboratory research as a sideline to other interests; not-for-profit organizations such as the Jackson Laboratory; and government research organizations such as the National Institutes of Health. Conditions of availability and motivating philosophies differ among them.
If investigators produced and distributed mice themselves, they would retain control over distribution, but they would also individually carry the burdens of the monetary costs of maintaining colonies and other infrastructure and of the time diverted from research. Such a distribution system would also preclude the kinds of potential economies of scale that are associated with large operations such as commercial enterprises or a national repository. However, there would presumably be a minimum of legal restrictions on the distribution and use of mice, costs for recipients might be lower than those associated with the use of commercial firms, and new strains could be distributed relatively quickly after they were created.
Additional disadvantages and uncertainties are associated with a distribution system that relies on individual researchers. Cage costs vary enormously from one academic institution to another, and they can be very high at some institutions. There would be problems of maintaining colonies of sufficient size to satisfy requests. Important demands would be placed on the time of technicians attached to an investigator who has agreed to provide an individual mouse.
Costs might be covered by obtaining subsidies from NIH, by charging recipients, or by having the investigator's institution cover the costs and receive payment from the recipients. Investigators could also offer the resource to a commercial firm under licensing agreements that could provide for distribution to other research workers at low fees.
However, major logistical problems are also associated with breeding, quality control, disease prevention, and regulations. Many of the strains require specialized breeding. At the Jackson Laboratory, many of the mutants are maintained by ovary transplantation, whereby mutant ovaries are placed into phenotypically normal mothers to provide a normal environment in which embryos can grow. There are problems with cryopreservation, packing, shipping, and invoicing–all things that an ordinary research laboratory is not set up to do.
A major problem is ensuring the quality of the mice that are distributed. The individual investigator may not pursue the same quality control that a commercial organization or a national repository might pursue to survey the animals that are being sent out continuously and to make sure that the transgene is still altered in the correct fashion and that the animal is free of pathogens.
Perhaps the most serious problem with this option has to do with the spread of disease between research colonies in the United States. For example, about 75% of the mice that the Jackson Laboratory import either are actively infected with a mouse virus or are serologically positive for antibody against viruses. At least half the mice that come in show evidence of prior hepatitis virus infection. Multiple distribution to research laboratories all over the
country could lead to a pandemic of mouse virus infections and seriously jeopardize biomedical research throughout the country.
For those reasons, relying on individual investigators might not be preferable. Furthermore, although many researchers are willing to share their resources, few are in a position to be or would wish to be seen as a primary resource for the distribution of laboratory animals.
Companies that maintain and distribute laboratory animals must, as a good business practice, produce a high-quality product and make it conveniently available (see Box 4.2 for two examples of firms that produce transgenic mice). They have the resources to develop pathogen-free, well-validated animals. They can take advantage of economies of scale.
However, they typically catalog only a few high-volume animals, and there would be little incentive to stock the numerous transgenic mice that might be used in small numbers. Costs of different strains might vary substantially: “boutique” mice (rare, highly specialized strains with low market demand), if stocked, might cost much more than more commonly used strains.
Box 4.2: Commercial Enterprises
Two companies, GenPharm International and Charles River Laboratories, can serve as examples of how for-profit organizations distribute genetically altered mice, some of the problems such enterprises encounter, and whether they can meet the needs of the academic research community.
GenPharm International, a small biotechnology company funded by venture capital, is based on transgenic science. One group of company scientists, in Europe, is developing transgenic cattle for the production of pharmaceutical proteins in cow's milk. Nutritional and therapeutic substances are potential products. The main goal of a second group of company scientists, in the United States, is to produce a mouse that will have the ability to produce fully human monoclonal antibodies.
Although GenPharm's main focus is on developing pharmaceutical and nutritional products, it has also become involved in supplying transgenic mice to the community. The latter line of business is important to GenPharm, according to David Winter, the company president, for “our visibility and our future activities–to maintain as close contact as we can with the field, so that we know what is going on in the field and can be very much a part of that field as we develop our own proprietary, positions. ”
The company sees its role as serving the scientific community with the introduction of a few selected transgenic models. Benefits of the service
include accelerating the development of these transgenic models, broadening their distribution, and providing a superior product to researchers so that they do not have to get involved in complicated husbandry issues. The company considers itself as playing a role in transferring technology from the academic laboratory to user communities and helping universities to derive a source of support for their research programs through commercialization of their inventions and discoveries.
GenPharm has required substantial upfront investment for the introduction of each transgenic strain to its product line. To cover licensing fees and expenses for cesarean derivation, colony expansion, and genotyping, an investment of approximately $90,000 is needed for each transgenic strain before the first shipment of an animal.
Two types of agreement are used in the distribution of the animal models: use agreements and cross-breeding agreements. Use agreements are similar to those imposed on a software buyer who, in effect, is “leasing” rather than buying the software. Cross-breeding agreements cost $1,000 per year per strain for academic-environment purchases and somewhat more in the industrial environment. Purchasers under the agreements may breed the animals and use them as they will under the usual constraints of required material-transfer agreements. Reach-through agreements are not requested. The comparatively high annual fee for academic researchers has caused some concern in the research community.
Currently, there are 110 signed agreements altogether They represent 70 institutions. Forty-seven of these agreements are with academic or nonprofit institutions. There are 15 cross-breeding agreements, mostly with academic institutions.
Charles River Laboratories is a major distributor of laboratory animals to academic and industrial users for research and testing purposes. It decides on scientific and business grounds whether it will accept a model offered to it by an academic investigator. Because of the large number of different mice being developed, there is need for a means to choose among them to determine which to breed and market.
Charles River has not yet found a transgenic rodent that is widely useful for pharmaceutical testing and development. What maintains its interest is the potential that a highly successful transgenic mouse model will be developed. As Glenn Monastersky of Charles River said, “We are in the business of supplying sophisticated, highly developed mice to people, and transgenics is supposedly the next phase of rodent use in R&D.” As for now, the firm sees itself as providing a service to academe and industry by making transgenic mice available.
However, individual investigators whose strains were managed by the commercial handlers of the mice would be relieved of the task of maintaining and distributing their strains.
Commercial firms might require purchasers to sign agreements governing use, cross-breeding, or other matters. These agreements could depend on whether the purchaser were an academic scientist or another commercial firm.
Not-for-Profit National Repository
A not-for-profit organization serving as a national repository might combine some of the advantages of a commercial system and a system based on the efforts of individual researchers (see Box 4.3 for an example of a not-for-profit repository). It would relieve individual investigators of the burden in time and money of having to maintain and distribute mice. It would act as a service to the research community, accepting mice, ridding them of pathogens, breeding them, freezing stocks of embryos and eventually mouse sperm, and distributing the mice and embryos at or below cost. It could take advantage of the economies of scale associated with a large organization devoted solely or largely to the maintenance and distribution of the mice.
A national repository could be an entirely new entity or could be based at an existing organization that would already have the necessary facilities and expertise. Individual investigators might have more confidence in an existing facility operated by scientists who have been investigators themselves and might feel that it would be sympathetic to the tradition of sharing.
The repository would need some mechanism to oversee the genetic portfolio maintained by the center. Keys to success would be long-term, sustained commitment continued efforts to improve efficiency, and new preservation technology to keep pace with development in the transgenic technology that produces the mice in the first place.
Any national repository would require extra funding and facilities to maintain colonies. Support would need to come from such sources as NIH. If the repository is reluctant to accept animals under license agreements, investigators who offer their animals to the laboratory might have to forego potential profits to have their animals accepted.
National Institutes of Health
Since the 1970s, NIH has maintained an animal facility. Its catalogue lists about 350 stocks and strains of animals. Although the facility is intended primarily for the use of intramural investigators, animals are made available to all requesters, including commercial enterprises, at cost.
Box 4.3: The Jackson Laboratory
The Jackson Laboratory has been distributing mutant mice and specialized genetic strains for about 60 years. Whereas commercial distributors concentrate on a total of about two dozen strains, Jackson maintains about 1,700 mutants and strains either as live mice or as frozen embryos. In a recent year, about 1.5 million mice representing about 500 strains were distributed. The one restriction Jackson places on the use of animals is that they are not for commercial resale. Recipients may breed the animals for their own use.
Jackson believes that it communicates with the research community on a collegial basis and shares the same value system as the investigators that it works with. Its approach is to select the mice to breed on the basis not of profitability but of the importance of the strains to biomedical research. A survey of 280 journals over a period of 6 months has shown that 85% of the genetically defined mice reported on in papers in those journals were obtained from the Jackson Laboratory, although Jackson provides only 15-20% of the mice used in laboratory research.
The Jackson Laboratory has recently established an induced-mutant resource to accept and distribute genetically altered mice it receives from outside sources. The first two lines that they have distributed are the MIN (multiple intestinal neoplasia) mouse, which is the mouse homologue of the human disease familial adenomatous polyposis coli and the CF (cystic fibrosis) mouse. Jackson also distributes frozen embryos from its stock of 1,000 mouse strains. Financial limitations will restrict the number of new mutant mice imported per year to about 50.
In setting up the induced-mutant resource, Jackson has decided that it will not accept mice that have any licenses or restrictions on them for use or breeding for basic-research purposes. If licenses are required for commercial use, Jackson requires that they be negotiated directly between the provider of the mice and the commercial user. The laboratory has reluctantly accepted the necessity to provide royalties to some of the providers to ensure that it can provide particular mice to the research community.
With enlargement of its facility. NIH might itself serve as a national repository for genetically altered mice. However, such an expansion might be expensive. Alternatively, NIH might fund the startup of an extramural national repository that would ultimately run on funds from the distribution of mice and contributions from other major commercial and nonprofit users. One possible advantage of setting up an intramural NIH facility is that if any patented or licensed strain developed with NIH support is donated to the government, it would be owned by the government, which could distribute the strain freely to researchers without a licensing fee.
Under either option, there might be delays in the review of requests as in the experience of other NIH repositories for antibodies or serum samples.
None of the options discussed addresses all the needs or problems associated with the distribution of genetically altered mice. Individual investigators might provide the most rapid initial distribution of new strains, but costs and logistical problems severely limit the utility of this approach. Commercial firms might provide the most convenient and least expensive service for commonly used strains, but they might not be able to provide rare strains, and they might require restrictions on use of breeding to protect profitability. A national repository, whether nonprofit or government, could provide a wide range of mice conveniently, but it might not be able to provide all desired strains at reasonable cost and in a timely manner without substantial government support.
In fact, the numbers of strains potentially available could in the end be so large as to require more than one repository or the use of more than one of the above mechanisms. In any case, it would be important to have distributed facilities to hold duplicates so that strains that are difficult or impossible to recreate would not be completely lost because of fire or other disasters.
HANDLING THE PROLIFERATION OF STRAINS
The large number of strains that are being produced creates a serious logistical problem for research. Consider, for example, that Drosophila researchers can draw on no fewer than 30,000 mutant strains. What are the implications if mouse researchers begin generating thousands of strains of genetically altered mice? For a number of reasons, it is impossible to preserve all strains as breeding colonies. Not only the logistics associated with managing purebred colonies of large numbers of strains, but also difficulties with survival or fertility can seriously impede the availability of genetically altered mice.
Alternative mechanisms are clearly essential if strains are to be freely available for current and future research. Cryopreservation is an important option. More than 20 years ago, the first embryos were cryopreserved successfully–i.e., they resulted in the birth of live young. Since then, thousands of cattle, mice, rabbits, and human beings have been born as a result of the transfer of cryopreserved embryos.
Any strain of mouse can now be successfully cryopreserved. Furthermore, all embryonic stages of development can be successfully cryopreserved, from
fertilized ova to blastocysts. The various procedures that have been developed involve cryoprotective additives and cooling of the embryos under conditions in which the cells undergo almost total dehydration. Liquid nitrogen is used for long-term storage. Returning samples to normal physiological function involves thawing, warming and removal of cryoprotective additives.
Cryopreservation methods can now yield at least 75% survival. The procedure is reliable. A frozen embryo can almost certainly be stored successfully for hundreds of years without any deterioration. Embryos preserved in the twentieth century could conceivably be used for research in the thirtieth century. This capability provides an important avenue for performing retrospective analysis.
The embryo bank at the Jackson Laboratory has cryopreserved more than 950,000 embryos of 1,100 strains. Of mice now held at Jackson, 510 strains exist only in cryopreserved form. Jackson routinely re-establishes more than 50 strains a year from cryopreserved embryos. Such cryopreservation need not be done only by institutions like Jackson. Individual investigators could consider the option of cryopreserving small numbers of embryos, which can be easily done with small capital investment. Investigators who had many different lines of transgenic mice in colonies could, with little expense and effort, start freezing their embryos to prevent loss by genetic or microbial contamination.
Cryopreservation of embryos, although effective, is not the most efficient means of preserving genetic material. Cryopreservation of sperm from genetically altered mice would require far fewer animals to provide a suitable stock of material for reconstituting the strain. Small aliquots of frozen sperm could be used to fertilize eggs in vitro or to inseminate females artificially.
The first mammalian sperm were frozen successfully almost 45 years ago. Since then, millions of cattle and thousands of human beings have been conceived by artificial insemination with cryopreserved sperm. But the same is not true for mice.
In the last couple of years, there have been reports, primarily from Japan, of the successful cryopreservation of mouse sperm. Cryopreserved sperm have yielded live young, especially when used for in vitro fertilization of oocytes, but the procedures now used for mouse sperm are empirical. The yield in general is rather low, and, most important, survival tends to be highly variable.
A major obstacle to the successful cryopreservation of mouse sperm has been the lack of research. Recent increased support for such research by NIH should help to overcome that obstacle. Also, a national repository could conduct research on ways to freeze, store, and use mouse sperm and eggs and methods to improve the efficiency of cryopreservation of embryos. Other possibilities are currently being explored, such as the preservation of intact ovaries and testes and cryopreservation of large numbers of samples on
magnetic strips. Huge data banks of genetic resources in the form of cryopreserved samples might be achievable in the near future.
Managing Data about Strains
Thousands of strains of transgenic mice and targeted mutations will almost certainly be generated by researchers over the next several years. It will be essential to keep track of them so that investigators can learn of the existence of a strain and of its characteristics.
The explosion of reported studies that use transgenic animals and, more recently, targeted mutation has made it nearly impossible for an investigator to review the entire field and maintain an awareness of the work going on an international basis. TBASE, a computerized database that stores and organizes data on transgenic animals and targeted mutations, was developed to fill that need. TBASE was initially supported with startup funding from the National Institute of Environmental Health Sciences and is now supported by the Department of Energy. It is operated by the Welch Laboratory of Applied Bioinformatics at the Johns Hopkins University School of Medicine in association with the Human Genome Data Base. The director of the laboratory and principal investigator for TBASE is David T. Kingsbury.
TBASE contains information pertaining to the experimental methods and techniques used in developing the transgenic or targeted-mutation line, the nature of the genetic construction used, and a wide variety of features related to the resulting phenotype, if one has been observed. Both published and unpublished results are present, and the TBASE developers have an active program of soliciting direct submission of experimental data so as to be more comprehensive in their coverage of the field. TBASE has undergone several schema revisions since it began, but the current schema appears to capture this rapidly moving field fully, and it is expected to remain stable.
To accommodate the rich cross-links necessary in genetic databases, the current TBASE schema contains a number of links to other databases, including the Human Genome Data Base, Online Mendelian Inheritance in Man (OMIM), the Mouse Locus Catalog (MLC), the Mouse Genome Database (MGD), and Pigbase. TBASE accommodates extensive comments on each major database object and a complete reference to the corresponding contact investigator.
The latest schema implementation accommodates references to “related” mutant or transgenic lines (parental or derived) in the appropriate cases. TBASE accommodates the full Institute of Laboratory Animal Resources (ILAR) laboratory codes and unique descriptive names where ILAR nomenclature has not been implemented. TBASE's unique identifiers (accession numbers or UIDs) make linking TBASE to other databases possible in cases in which investigators wish to access the relational database version.
TBASE is implemented in a fully relational database-management system that is used for curation and for public access. For most investigators, the most convenient access is through Gopher, but TBASE will soon be accessible through the World Wide Web, which will also serve as a route of submission. The Gopher software is widely available and information about its acquisition can be obtained through the Welch Laboratory user support line at (410) 955-9705 or through email at firstname.lastname@example.org. Connectivity to the Johns Hopkins BioGopher is achieved by configuring the client to connect to gopher.gdb.org, port 70. For users who do not have access to computers on the Internet that have a Gopher client, access through a network Gopher client may serve as the alternative method. From a networked machine with standard network software, a client may be reached by typing telnet mouse.gdb.org (login: tbase, password: <return>). Finally, telephone dialup access is available at (410) 614-2665 (vtl00, 8 bits, no parity, 1 stop). When TBASE is fully implemented, by late 1994, it will be available in a full client-server SQL query-compatible mode and be integrated within the complex of genomic databases.
Data acquisition is effected by active literature-scanning and processing of direct submission forms. Literature-scanning refers to direct data extraction from the scientific literature through careful examination of a large number of journals, which have been statistically identified as periodically stable sources of such information. The data are later processed and manually entered into the database by TBASE staff. Active literature-scanning has so far been the primary mode of data accumulation and has ensured that the database accurately reflects the general progress of transgenic research.
The second method of data acquisition is the processing of direct submission forms, which consist of paper and electronic entry forms and templates. Appropriate paper entry forms have been designed for each of the three major TBASE object classes–Laboratory, DNA Construct, and Transgenic Line–closely following the format that is reflected by the recently revised schema. The paper forms may be obtained from the Welch Laboratory. The paper entry forms contain obligatory information (equivalent to the “not null” condition in the data dictionary); this includes contact information and any citations originally describing the founder lines or constructs, which must be clearly and fully provided as cross references. One complete set should be processed per transgenic animal or targeted mutant. Individual forms, however, may be used to modify or update information on a given line or mutant that already exists in the database.
The use of electronic submission forms and templates constitutes the final data-acquisition strategy, which is going to be implemented in the immediate future on satisfactory implementation of the revised schema. Original investigators and submitters will be able to use software submission tools with error-checking capabilities for more efficient and timely contributions to the database.