Future Directions in Research Animal Use: Infrastructure, Cost, and Productivity
The information in this chapter is based on the experience of the committee members and informal consultation with a number of investigators who use a considerable number of animals in their research. The purpose is to facilitate planning by projecting the likely expansion in the use of animals. Data contained in the US Department of Agriculture 's annual Animal Welfare Report show a decline in the use of all animals covered by the Animal Welfare Regulations over the last decade, from 1.75 million in 1989 to 1.2 million in 1998. The use of all species except nonhuman primates fell. However, rats, mice, birds, and all cold-blooded animals are excluded from coverage. It is estimated that over 90% of animals used in research are mice and rats. It seemed important to examine trends in the use of mice because it is the committee experience that such use will drive the need for new or renovated animal research facilities in the near future.
The major increase in animal research in the last few decades has involved the use of the mouse as an experimental animal. It is likely that the largest increase in demand for animal care will be for mice, although other experimental systems—such as flies, worms, fish, frogs, and pigs— are being further developed and used.
A number of factors influence the use of the mouse as an experimental system. A major initial factor was the development of transgenic mouse technologies in the middle 1980s. Use of transgenes to achieve
deregulated or tissue-specific expression of desired genes in mice was an important component of research that led to major breakthroughs in several fields of biology. In cancer research, expression of dominant oncogenes as trans-genes led to the development of basic and applied models for the study of a wide variety of neoplasms. The ability to achieve specific transgene expression has led to a large increase in newly generated mouse models and has resulted in a quantum leap in our level of understanding of the development and function of the immune system.
The first successful application of embryonic stem cell (ES cell)-based approaches to introduce gene-targeted mutations into mice was reported less than 10 years ago (Capecchi 1989). This technology has had an even more dramatic impact than transgenesis on basic and applied research, further establishing the mouse as a major experimental model system. Until several years ago, application of gene-targeted mutation technologies in mice was limited largely to a handful of major research centers or specialized investigators. However, as with most technologies, gene-targeted mutation approaches and reagents have been refined to the point where they are now accessible to most research institutions and are readily used by much of the biomedical research community. This technology for defining mammalian gene function in a physiologic setting, unimaginable 20 years ago, has become one of the most widely applied and most informative tools of biologic research.
Application of gene-targeted mutational analyses is likely to continue to increase demand for the mouse as a model system in the next decade, especially when coupled with powerful new technologies—such as genomics —and the potential power of combinatorial studies of existing or future targeted mutations.
FACTORS CONTRIBUTING TO INCREASED MOUSE USE
The Genome Project and functional genomics, including gene-mapping experiments and gene-function validation, are major factors that will increase the use of mice. The project has rapidly increased the volume of known genetic sequences and identified genes, a large proportion of which have unknown functions. These sequences are being made available in easily accessible genomic databases—leading to more target sequences for gene-targeted mutation. The use of mice for large-scale gene mapping experiments and functional genomics will increase dramatically as these mutagenesis projects get under way. The largest increase in animal use will presumably occur mainly in a small number of large centers and in industry, but the overall impact will be widespread.
Gene identification will become progressively easier as better mouse genetic maps are constructed, although this will lag a few years behind
human maps. In the meantime, as the human map nears completion, information from syntenic regions in the mouse might be useful and speed up gene identification based on mapping information. That in turn will lead to use of animals for validation of function. Increased numbers of genes identified through the Genome Project could potentially lead to thousands of new gene-targeting experiments, provided that resources continue to grow.
Developing technologies, such as array analysis, will increase the utility of mouse models. These powerful diagnostic techniques will enable analysis of expression patterns in, for example, tumor models that express a variety of genes in the same pathway. As techniques become more sophisticated, it will be possible to look at early disease stages and to dissect complex interactions in tissues. In addition, gene chips and protein chemistry will require an increased number of animals to generate proteins for analyses.
There are increased interinstitutional transfers of novel lines coupled with combinatorial interbreeding of different lines that will lead to increased use of mice. As an example, some 300 mutant lines were brought into the Dana-Farber Cancer Institute (DFCI) and 300 other lines were sent out of the DFCI in the last year. The ready transfer of lines, coupled with interbreeding of mutant and transgenic lines to generate large numbers of new lines, will result in a large increase in the number of mice used. Examples of the application of interbreeding of lines include:
Generation of animals with polygenic mutations, using multiple mutant or transgenic backgrounds for basic studies in such fields as cancer biology, immunology, and neurobiology. The combinatorial breeding of different mutant backgrounds could generate huge increases in numbers of experimental mice.
Genetic-modifier studies, for example, analyses of favorable and adverse influences of genetic background on current or future cancer-model strains.
Polygenic disease models involving multiple contributing genetic loci with respect to such diseases as cancer and some immune diseases.
Back-crossing and inbreeding to create the desired genetic backgrounds for immunology studies.
Conditional targeted mutations and tissue-specific mutations (tet, cre/lox, and other similar strategies) will further increase animal use for modeling and developmental studies. The technology is still being developed, and it will be a few years before it sees widespread use. Rapid improvements could occur if National Institutes of Health (NIH) or foun-
dation resources are targeted to improving and distributing this technology.
Chemical and viral mutagenesis of mouse germline will be used to study environmental mutagenic effects, to identify new genes involved in development and cancer, and to create models for therapeutic trials. Interest in transgenic mice has the potential to increase dramatically. In many instances, well-designed transgenic experiments, potentially in combination with knockouts, can be more informative. Therapeutic models—for example, for cancer therapy, gene therapy of genetic diseases — are expected to increase.
As basic understanding of molecular biology increases, there will be an increasing interest in and emphasis on whole-animal in vivo experimentation. This will increase the use of mice for experiments involving gene transfer into preimplantation and postimplantation embryos and observations of the effects in organ culture and in utero.
The ease of mouse-genome manipulation resulting from the establishment of core laboratories for generation of mutant lines, histopathologic analyses, genotyping, and other analyses will benefit the national genomics initiative if creating these core laboratories becomes a national priority.
An increase in NIH monetary support for infrastructure development and the payment of direct costs could determine the level of animal use. Many institutions are pursuing the construction of new animal space and space renovation for modernization. If the national economy stays robust, the NIH budget should grow and make resources available to continue expanding mouse work. Growth of the infrastructure portion of the National Center for Research Resources budget of the NIH has not kept pace with the need for new animal research space.
New design concepts and technologies are resulting in more efficient and larger animal facilities, which have greater capacity. Many institutions now regard the capacity of their animal facilities as the major factor that limits the expansion of their biomedical research programs.
POTENTIAL STRATEGIES TO DAMPEN THE EXPLOSION IN MOUSE USE
Because of the advances noted above in the use of the mouse as a primary model system for the investigation of mammalian genetics, it is inevitable that the number of mice used in institutional research programs will continue to surge. On the basis of the committee's experience, several useful strategies are suggested to manage growth of mouse populations:
The use of prudent colony management—especially involving breeding animals, effective database management, and accelerated genotyping—can reduce generation and retention of extraneous animals. These colony-management techniques could stimulate other choices for institutions that might choose to use external specialists with relevant expertise to establish training programs to address specific needs.
Preservation of lines, embryo freezing, sperm cryopreservation (the least expensive method, pending resolution of issues related to pathogen transmission and long-term viability), and viable in vitro fertilization methods might reduce the need to maintain various mouse mutants as active populations in facilities.
The use of satellite or centralized animal research facilities might reduce the overall impact on an institution's resources if there are financial incentives to house off-site in commercial contract sites.
More central repositories for unique mutants are created to meet the higher demand for mutants.
Alternative central animal research facilities are created through regional consortia or independent academic medical centers with outstanding histories of laboratory animal management.
Improved animal research facilities are provided that can result in better health of strains and less need for strain re-derivation or regeneration after disease outbreaks or other cataclysmic events.
Centralized cores for common strains, such as cre/lox and RAG, might reduce overall numbers as investigators become confident about timely strain availability and effective strain distribution.
In some areas, the mouse might be replaced in genetic studies with simpler organisms that have sufficient homology (such as yeast, Drosophila, and Caenorhabditis elegans) as a result of genome-sequence determination, but this effect is probably transitory.
SUMMARY OF MOUSE PROJECTIONS
Barring a major decrease in funding, factors that support a substantial increase in use of mice greatly outweigh factors that would decrease their use. Many institutions have projected a threefold increase over 5 years, assuming that space and funding are adequate, but some suggest that such a projection is very conservative. Lower estimates from other institutions (including Harvard and Albert Einstein) might reflect the constraints on space that these institutions encounter.
POTENTIAL FOR USE OF OTHER TRANSGENIC SPECIES
Rat and Rabbit
Technologies have been developed for generation of transgenic rats and rabbits. The use of transgenic rats and rabbits also occurs in academic settings, although this will depend even more heavily on funding because such models are potentially very expensive. Support for these models will depend to some extent on the technologic ability to make physiologic measurements or conduct disease interventions in these animals that cannot be carried out in mouse models.
Transgenic pigs are more attractive than mice for modeling human vascular diseases and, potentially, organ transplantation. The use of this animal model system in translational research is substantial in the academic setting.
Other Transgenic Mammals and Birds
The application of transgenics or gene-targeted mutations in other large animals or birds could also increase but would probably find most current use in applied science in commercial settings. With the exception of pigs and nonhuman primates, there is no obvious reason to expect an increased demand for large animals in research over the next 5 years.
Some growth in use of Xenopus is expected. NIH is considering a plan to initiate a genome project for frogs that involves expressed-sequence tags, using Xenopus tropicalis for frog genetics. Frogs have been used traditionally for developmental and cell biology studies.
The use of zebrafish as a model for studying development has shown a high degree of promise. Zebrafish require relatively low maintenance. Large-scale mutagenesis screens for recessive traits have been successfully carried through to identification and cloning of mutant genes. Those chemical mutagenesis screens have been successful in isolating zebrafish lines that contain mutations affecting organogenesis and neurogenesis, physiologic function of such organs as the heart and a variety of muta-
tions affecting different stages of embryonic hematopoiesis. Most of these mutants live well beyond the stages of early development and so allow identification, propagation, and genetic characterization.
The use of zebrafish for the study of vertebrate embryonic development, neurogenesis, organogenesis, medically relevant pathophysiology, and fundamental mechanisms of cancer might increase exponentially over the next decade. Over the last year, an NIH-sponsored zebrafish genome initiative has been launched and has resulted in a vast improvement in knowledge of the genome of this organism. Large regions of synteny have been identified in the mouse and the human; this indicates that advances in genomic sequencing in these species will also facilitate use of the zebrafish model.
In summary, the major findings and opinions expressed in this chapter are as follows:
The Human Genome Project and functional genomics supported by a diverse array of experimental approaches will continue to fuel the use of the mouse as the primary experimental model system in the investigation of mammalian genetics.
Many strategies may prove to be useful to hedge the ongoing explosion in mouse use. These include: improved colony management; database management; techniques to maintain genetic stocks without maintaining active populations; consolidation of key mutant lines or strains into fewer facilities to eliminate redundant production while maintaining prompt distribution; and continued animal health improvements and the replacement of mice with simpler organisms when applicable.