Microbial and Phenotypic Definition of Rats and Mice: Proceedings of the 1998 US/Japan Conference (1999)

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The Need for Defined Rats and Mice in Biomedical Research: Problems, Issues, and the Current State of Affairs Norikazu Tamaoki Professor, Department of Pathology, Tokai University School of Medicine Kanagawa, Japan GLOBAL HEALTH ISSUE AND THE NECESSITY OF LABORATORY ANIMALS Speaking on behalf of the Liaison Committee for Laboratory Animal Sci- ence of the Science Council of Japan, I am pleased to discuss laboratory animals from the viewpoint of global health issues. My talk will be rather general and will include the following three major topics: (1) importance of laboratory animals for human health, (2) laboratory animal models in major disease categories, and (3) aspects of future laboratory animal use. IMPORTANCE OF LABORATORY ANIMALS FOR HUMAN HEALTH Many aspects of health issues are directly related to the socioeconomic status of the world’s regions and countries. In the populations of developing countries, nutrition and infection are urgent problems to be solved. However, in the devel- oped countries, life-style diseases are important issues. Emerging and reemerg- ing infectious diseases and drug abuse are important in both developing and developed countries. Risk factor analysis shows that 50% of all types of disease is due to life-style, 20% to environmental factors, 20% to genetic factors, and the remaining 10% to medical care. To prevent and treat such illnesses, it is necessary to understand the mechanism of diseases and to develop intervention systems including medical care. New drugs and health education are indispensable for disease prevention. 7 

8 MICROBIAL AND PHENOTYPIC DEFINITION OF RATS AND MICE For this purpose, we need more accurate models and assay systems for both normal and abnormal human conditions. Recent results of experimental gene therapy against tumor angiogenesis serve as an important example of the necessity of a whole organism model. It has been documented that growth of human cancer in vivo is dependent on vascularity and blood supply. We have shown that one of five isoforms of vascular endothelial growth factor—VEGF 189, a potent vascular growth factor—is responsible for growth and metastases of human cancers including colon (Tokunaga and others 1998), lung (Oshika and others 1998), and kidney (Tomisawa and others 1999). As a model for cancer gene therapy, transfection of the ribozyme that specifically catalyzes VEGF 189 into a cancer cell line has little effect on cell growth in vitro. In contrast, the same procedure inhibits in vivo human tumor growth xenotransplanted in severe combined immunodeficiency disorders (SCID) mice by suppressing angiogenesis (Oshika and Nakamura, manuscript in preparation). Clearly, to study the complex function of the organism’s multicellular or multi- organ system requires use of a whole organism (namely an animal) model. Accumulating data on human and mouse genomes and advances in gene technology have enabled us to have a more accurate understanding of gene struc- ture and function. However, with preliminary results obtained from genetically engineered mice, we are still far from our goal of understanding the function of the whole organism. Knockout or transgenic mice that have been developed represent the change in only one or a few among 100,000 genes in the whole genome. Gene function is not uniformly expressed in cases involving alternative splicing or other mechanisms resulting in production of several isoforms of gene products with different biological activity. In addition, evaluation of a cancer gene therapy model requires not only cancer cells, but also supporting tissue. LABORATORY ANIMAL MODELS IN MAJOR DISEASE CATEGORIES Major disease categories for which appropriate animal models are needed include infectious disease, immunological disease, cancer, and life-style diseases. Details about these disease categories follow. Infectious Disease Elucidation of receptor molecules for microorganisms and toxins enables us to change the ordinary host range and to develop animal models susceptible to various human-specific pathogens. Polio virus-susceptible mice produced by CIEA are a good example. Additional studies of virus receptors and coreceptors will hopefully create various models for human infectious diseases. Animal models for parasitic disease is very important, but few practical models exist. 

NORIKAZU TAMAOKI 9 Recent studies on cytokine reaction patterns disclosed TH1- and TH2-type immune response. These results are helpful for studying the mechanisms in- volved in leishmaniasis and schistosomiasis (Mosman and Coffman 1989; Wynn and others 1995). In addition, progress in the development of new vaccinations such as DNA vaccine has been made through studies with laboratory animals. Immunological Disease Many types of immune-deficient models have been useful for human to mouse tissue xenotransplants. SCID mice and Rag2 KO mice are also interesting from the standpoint of genetic instability. Autoimmune models have a long history starting from the study of mouse genetics in mutants. Allergy models have been developed in many institutions. In addition, the study of cell adhesion molecules in transgenic and knockout mice has stimulated progress in the study of inflammation and in understanding cell behavior in vivo. Cancer Genetically engineered mice have played an important role in cancer research results based on the accumulation of data related to prenatal and postnatal gene abnormalities. Application of these data to angiogenicity assays appears to be a very promising biomedical tool for cancer treatment and prevention. The effects of background genes are also very important for our understanding of metabolism of carcinogens and organ-specific development of tumors. Life-style Diseases The life-style disease category includes major diseases in developed coun- tries, such as cardiovascular disease and diabetes. Considerable progress has been made in the field of cardiovascular disease, including hypertension and atherosclerosis, using transgenic mice expressing human renin-angiotensin gene and scavenger receptor gene. Here again, the whole body model is very impor- tant in understanding pathophysiology of disease due to multiple gene errors superimposed on human life-style factors. Risk Assessment for Environmental Factors Laboratory animals are important for the assessment of environmental risk because they serve as whole organism models for many risks of unknown etiol- ogy. The models are used not only for drug testing, but also for assay systems to evaluate various risks to human health. Selecting appropriate models for specifi- cally targeted risks is extremely important. 

10 MICROBIAL AND PHENOTYPIC DEFINITION OF RATS AND MICE ASPECTS OF FUTURE LABORATORY ANIMAL USE For our common goal—human health—we must develop and produce objec- tively oriented and quality-controlled laboratory animals. Laboratory animals used in the future should be purposefully selected for particular studies, be of reasonable cost both monetarily and in terms of life expectancy and maintenance, and be easily available. Although we currently have many disease models for biomedical research, most of the animals used for specific purposes do not fulfill the criteria described above. We need to develop a specialized support system to supply future laboratory animals. Such a system should be based on interdisciplinary research (including gene technology), biological databases, the entire field of human health science, novel methods of reproduction and development, and a system of animal care based on laboratory animal science. In conclusion, I would like to propose the establishment of new collaborative networks to unite government, academia, and industry for the development of the future of laboratory animals and laboratory animal science. QUESTIONS AND ANSWERS C. ABEE: With regard to your comment about the collaboration of govern- ment, industry, and academia, could you please provide your perspective on how that is done in Japan? N. TAMAOKI: I think there is no such system in Japan. Individual insti- tutes such as CIEA conduct it; however, as Dr. Gill pointed out, laboratory animal technology is progressing rapidly and it is too expensive to keep them in one institute. I believe that we need more united resources from governmental budgets and industry monies to maintain standardized, high-quality animals. I would also like to mention the pitfalls that exist with laboratory animals as a result of transgenics. There is a great difference between such genetically engi- neered animals and the reliability and availability of other laboratory animals. For this reason, we need a bridge between research models and laboratory ani- mals, which ideally would be supported by government and industry. If Japanese and US governments collaborate on this point, it would be better for both coun- tries. Unfortunately, we do not have a real system at this time. The most important issue right now is how to define the phenotype of ani- mals. It is very important to bridge the gap between genotype and phenotype. In the research field, it is very important to study the expression and mechanisms of genes. Gene expression is controlled in gene products of other genes. There are networks or cascades of functional products of genes, but at present, we do not have enough methods to check such a process, which takes time. For the moment, I think the practical way is to create a new method for defining the phenotype of animals. The functional phenotype must be defined by the reaction of animals to 

NORIKAZU TAMAOKI 11 some standard substance depending on the objectives of the experiment—for a metabolism study, for a neurology study, and so on. REFERENCES Mosman, T. R., and R. L. Coffman. 1989. TH1 and TH2 cells: Different patterns of lymphokine secretion lead to different functional properties. Ann. Rev. Immunol. 7:145-173. Oshika, Y., M. Nakamura, T. Tokunaga, Y. Ozeki, Y. Fukushima, H. Hatanaka, Y. Abe, H. Yamazaki, H. Kijima, N. Tamaoki, and Y. Ueyama. 1998. Expression of cell-associated isoform of vascular endothelial growth factor 189 and its prognostic relevance in non-small cell lung cancer. Int. J. Oncol. 12:541-544. Tokunaga, T., Y. Oshika, Y. Abe, Y. Ozeki, S. Sadahiro, H. Kijima, T. Tsuchida, Y. Yamazaki, Y. Ueyama, N. Tamaoki, and M. Nakamura. 1998. Vascular endothelial growth factor (VEGF) mRNA isoform expression pattern is correlated with liver metastasis and poor prognosis in colon cancer. Br. J. Cancer 77:998-1002. Tomisawa, M., T. Tokunaga, Y. Oshika, T. Tsuchida, Y. Fukushima, H. Sato, H. Kijima, H. Yamazaki, Y. Ueyama, N. Tamaoki, and M. Nakamura. 1999. Expression pattern of vascular endothelial growth factor isoform is closely correlated with tumour stage and vascularization in renal cell carcinoma. Eur. J. Cancer 35:133-137. Wynn, T. H., D. Jankovic, S. Hiney, A. W. Cheever, and A. Sher. 1995. IL-12 enhances vaccine- induced immunity to schistosomiasis mansoni in mice and decreases T helper 2 cytokine ex- pression, IgE production, and tissue eosinophilia. J. Immunol. 154:4701-4709.