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Phenotype Assessment Requires More Than a Casual Observation Philip A. Wood Professor, Department of Comparative Medicine University of Alabama Birmingham, Alabama Genetic differences among animals can often lead to differences in pheno- type. Now that we are in the era of creating animals with planned genetic differences, particularly âgene knockoutâ mice, we often anticipate what their phenotypes will be. Frequently, however, there may be no abnormalities or there may be unexpected abnormalities resulting from the intended genetic change. Frequently there are unexpected interactions with downstream pathways whereby the genetic background can markedly influence the phenotype resulting from a specific genetic change. There also have been gene knockout mice declared as having no abnormal phenotype; but when subsequent more specialized analyses were completed, striking abnormal phenotypes were discovered. Not only will genetic background significantly affect the phenotype of any given gene muta- tion as discussed by others at this meeting, but common environmental influences such as diet or cryptic infectious disease may also have a profound influence on the overall phenotype. The goal of this paper is to discuss a general approach for carefully assessing the many important influences on phenotype that are not often readily apparent at first glance. It seems to me that the issue of phenotyping genetically altered animals is so complex, and subject to so many subtle factors within the animal as well as its environment, that we must begin thinking in terms of paradigms. I describe here a paradigm to consider when approaching phenotype assessment of mice and rats. This paradigm is offered as an approach undergoing further refining as our assessment tools improve. I have divided this systematic approach into primary and secondary levels of assessment for the simple reason that all possible analy- ses are not practical for any animals. Additionally, the primary level assessment 58
PHILIP A. WOOD 59 can and should be available for investigators at most biomedical research institu- tions, but the secondary level of assessment will likely require more specialized expertise and equipment and could be integrated into nationally based networks established for phenotype assessment. Both components will be crucial for fully assessing phenotypes and fully using the vast number of rodent models currently being developed and studied. PRIMARY LEVEL ASSESSMENT: FIND ABNORMALITIES The goal of the primary assessment is simply to find abnormalities, through the following: 1. Clinical Assessment: Many knockout mice are initially on C57BL/6 Ã 129/Sv hybrid background, therefore controls should be littermate controls with a similar mixed background. a. Litter size: number born/weaned, sex, and genotype distribution b. Visual observation, particularly during the dark cycle when rodents are most active. Observe for behaviors that are aggressive, hyperactive, hypoactive, and so forth c. Observe for any coat color differences, skeletal or other body confor- mational changes, and failure to thrive. 2. Pathologic examination: Recommend evaluating both weanlings and retired breeders a. General necropsy to observe for any gross lesions and histopathology of all organs by an experienced rodent pathologist b. Microbiologic/serologic/parasite evaluation to detect any background infectious disease that may confuse the phenotype resulting from a gene mutation. c. Clinical pathology measures such as blood counts and simple urine analysis for protein and glucose. d. Determine life span and reevaluate phenotypes in old age. SECONDARY LEVEL ASSESSMENT: EVALUATE AND QUANTIFY ABNORMALITIES The goals of the secondary assessment are to evaluate and quantify the abnor- malities found during the primary assessment. This will often require more specialized expertise and technology. 1. Embryologic evaluation a. If abnormal litter size and genotype distribution are observed, these animals should be evaluated for gestational loss versus neonatal loss.
60 MICROBIAL AND PHENOTYPIC DEFINITION OF RATS AND MICE This evaluation often requires timed matings with careful embryologic evaluation to detect the specific gestational stages when the animals die. 2. Specialized pathologic evaluation a. Specialized stains for lesions detected by standard workup b. Electron microscopy for cellular lesions that are not discernable at the light microscope level c. Further evaluation of any blood cell count abnormalities with FACS analysis of leukocytes and other more specific immunologic measures d. Specialized organ assessment such as specialty pathologic evaluation of heart changes, eye changes, bone changes, analyses of neuron/ neurotransmitter distribution, and so forth. 3. Specialized biochemical analyses a. Metabolite analyses on blood, urine, tissue extracts for specific metabolites such as amino acids, lipids, carbohydrates in deficient or excessive concentrations. These assays may require very specialized equipment and expertise with small sample size. b. Enzyme or other specific protein analyses. This analysis would include not only assays that demonstrate the presence or absence of a protein, but also functional assays that may be crucial for corroborating any abnormal metabolite assays or blood cell abnormalities. c. Hormone analyses. This analysis can be particularly important in diabetic animals as well as those with failure to thrive, small body size, infertility, skeletal abnormalities, behavior abnormalities, or skin disease. 4. Physiologic assessment a. Pathologic evaluation may indicate organ dysfunction such as hyper- plastic or hypertrophic enlargement, atrophy, or absence. Technologies are being developed to more thoroughly assess physiologic function such as miniaturized equipment that can transmit data via telemetry for these valuable physiologic measures in the awake unrestrained animal. Minia- turized instrumentation for procedures such as ultrasonography, mag- netic imaging, DEXA analyses, indirect calorimetry studies, and other such devices are becoming increasingly available for these specialized measures in rodents, including those that are especially difficult in mice. 5. Behavioral assessment a. This is an important, developing area of biomedical research that will take advantage of the numerous genetic modeling approaches provided by rodents. There already are many behavioral differences observed among the inbred strains of rodents. With the gene mapping tools now available many genotype/phenotype correlations can be pursued includ- ing studies pursuing the genetic components of drug abuse and mental illness. There are knockout mice that also have abnormal behavior that need evaluation. This will require not only the current behavior assess-
PHILIP A. WOOD 61 ment paradigms, but also specialized physiologic assessment with loca- tion specific brain implants and EEG type measures. 6. Pathologic effects a. Rodents have a wide range of susceptibilities to common laboratory pathogens, many of which alter biologic responses (NRC 1991). This susceptibility has been well documented for several infectious agents, pointing out at least two issues to consider: (1) To fully evaluate the phenotype of a new model, it should be documented free of these patho- gens that may induce unwanted phenotypes. (2) For genetic manipula- tions involving immune functions and related effects, the animalâs sus- ceptibility to even opportunistic pathogens may markedly influence the phenotype. Thus, when evaluating the phenotype with a specific in- tended effect on these systems, this potential influence must be carefully controlled for and assessed. ENVIRONMENTAL INFLUENCES 1. Dietary changes and unsuspected constituents or deficiencies in diet may play important roles in expression of phenotypes. Although many rodent diets appear fairly similar, subtle changes in constituents can have significant effects on the animals. Some examples include the possible roles that phytoestrogens, found in soybean-based protein, may have in masking gender-specific pheno- types. Likewise, studies involving blood pressure evaluation may be signifi- cantly affected by simply changing rodent diet vendors who supply different quantities of salt in their diets. 2. Significant alterations in reproductive phenotype can result with major changes in animal room temperature, humidity, pheromone effects, and noise. EXAMPLES 1. BALB/cByJ versus BALB/cJ mice. This example will illustrate the drastic behavioral differences and metabolic differences in mice that appear very similar at first glance (Wood and others 1989). 2. Male-specific heart changes seen in mice with long-chain acyl-CoA dehydrogenase deficiency. A concern is that the soy-based protein in the diet consumed by these mice may mask the net cardiac changes seen in this model (K. B. Cox, D. M. Kurtz, and P. A. Wood, unpublished results). 3. Strain specific responses to Mycoplasma pulmonis infections in mice (Cartner and others 1996). 4. Examples of the unsuspected changes in arginine metabolism in rat models used in studies of salt-sensitive hypertension (Wood and others 1998).
62 MICROBIAL AND PHENOTYPIC DEFINITION OF RATS AND MICE In summary, phenotypic assessment is an important part of the genotype/ phenotype correlations that we are all interested in understanding with mutant rodent models in biomedical research. It is important to follow a systematic approach for the assessment, so that the model can be used to the fullest extent. Considering the problem of trying to evaluate the myriad of effects resulting from a single genetic change is a daunting task. The goal of this presentation is to provide a framework to consider this important problem. REFERENCES Cartner, S. C., J. W. Simecka, D. E. Briles, G. H. Cassell, and J. R. Lindsey. 1996. Resistance to mycoplasmal lung disease in mice is a complex genetic trait. Infect. Immun. 64:5326-5331. NRC [National Research Council]. 1991. Infectious Diseases of Mice and Rats. A report of the Institute of Laboratory Animal Resources Committee on Infectious Diseases of Mice and Rats. National Academy Press, Washington, D. C. 397 pp. Wood, P. A., B. A. Amendt, W. J. Rhead, D. S. Millington, F. Inoue, and D. Armstrong. 1989. Short- chain acyl-coenzyme A dehydrogenase deficiency in mice. Pediatr. Res. 25:38-43. Wood, P. A., D. A. Hamm, P. Y. Chen, and P. W. Sanders. 1998. Studies of arginine metabolism and salt-sensitivity in the Dahl/Rapp rat models of hypertension. Mol. Genet. Metab. 64:80-83.