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

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Genetic and Phenotypic Definition of Laboratory Mice and Rats / What Constitutes an Acceptable Genetic-Phenotypic Definition Hideki Katoh Chief, Genetics Division ICLAS Monitoring Center Central Institute for Experimental Animals Kanagawa, Japan GENETIC STUDIES ON CLOSED COLONIES OF THE RAT The following questions are important to all of us who are interested in human racial differences: • Are the ancestors of present races common or different? • How do we understand genetic relations among the races? Population genetics studies provide answers to these questions as follows. HUMAN ANCESTRY We know that all human races have one common ancestor, whose progeny migrated across the world roughly 50,000 to 200,000 years ago. After these common ancestors settled in various areas and adapted to the environments, they developed as races. That today’s races have a common ancestor means that they had common genes. These races exist as a result of accumulated genetic changes caused by gene mutations and of gene frequencies at many loci. Such genetic changes resulted in racial diversity, with different genetic characteristics such as skin color and height. 48 

HIDEKI KATOH 49 Genetic Relations Among Races Population geneticists have calculated gene frequencies of blood types and have shown similarities among races using differences of various blood type frequencies from blood group data of all of the races. Genes and their frequen- cies of common ancestors of the human races were randomly changed and varied when the ancestors dispersed to various parts of the world and settled there. Randomly occurring changes of gene frequency are called genetic drift. One example of genetic drift obtained by computer simulation can be seen in Figure 1. If the ancestors had A and B blood types at rates of 50% each, one of the two races will show a higher percentage of the A type and the other loses the A type after 300 generations. These changes are natural and occur by chance. Assuming a breeding cycle of 30 years, 300 generations is equivalent to about 10,000 years in humans. This time is sufficient to develop races different from the original one. However, it is important to remember that mating between races is possible, and such offspring will also be reproductive. This ability to produce fertile offspring means that no race has developed into subspecies. In the mouse, 1,000,000 years are required for genetic divergence to lead to a subspecies. Ethnic Differences It is interesting to consider the number of loci required to identify ethnic differences of human races. In Figure 2, the results for several racial groups can be seen using 12 loci, with Caucasoids, Mongoloids, Negroids, and Australoids clearly identified. These results agree with those of anthropology and cultural anthropology. What happens if we have fewer loci for an ethnic analysis? Figure 3 FIGURE 1 A computer-simulated result of genetic drift. 

50 MICROBIAL AND PHENOTYPIC DEFINITION OF RATS AND MICE NEGROID 49 29 50 AMERICANDS in North America 47 CAUCASOIDS 26 48 A 28 32 16 34 18 1 39 33 23 38 19 20 44 43 37 22 5 7 24 21 45 17 42 14 AMERICANDS AUSTRALOIDS MONGOLOIDS in South America FIGURE 2 Genetic relations among 31 races obtained from the gene frequencies of 12 loci. FIGURE 3 Genetic relations among 50 races obtained from gene frequencies of ABO, Rh, and MN blood types. 

HIDEKI KATOH 51 illustrates the results for the same racial groups using only three loci. As expected, the results are incomplete because some races are grouped into the wrong groups. Thus more reliable results are obtained in a population genetics study by using as many loci as possible. LABORATORY ANIMALS Closed Colonies To apply the genetic information described above to laboratory animals, it is necessary to define “laboratory animals.” Mice and rats include both laboratory and wild animals. Wild animals have been domesticated, and inbred strains and closed colonies have been bred as laboratory animals. In addition, there are many inbred strains and closed colonies with particular characteristics. As with humans, laboratory and wild animals also have common ancestors and so have inherited the same set of genes a long time ago. At the beginning of the 20th century, inbreeding was started to obtain inbred lines of mice. The oldest lines in the mouse have approximately an 80-year history. Because mice and rats reproduce three times (generations) a year, an 80- year history is equivalent to 240 generations. We can anticipate that two lines derived from a single ancestor differ genetically in the same way as two human races differ. It is important to remember that in spite of the two lines being separated 80 years ago, they can still mate and produce fertile offspring. However, in the case of closed colonies, mice and rats have approximately a 70-year history (Lindsey 1979; Morse 1981). Closed colonies also have several sublines derived from the main colonies. Genetic analysis reveals fewer differ- ences among closed colonies of mice and rats than among humans, indicating less genetic drift. One important problem exists in the case of laboratory animals, as illustrated in the following extreme example. Suppose that a colony of breeder A was supplied to breeder B 10 years ago. Genetic drift is sufficiently slow that we can assume that in 10 years, the two lines will not diverge dramatically. In fact, the two breeders do anticipate such changes. However, it is possible that breeding schemes can be accidentally mixed up by people working in animal facilities. It is possible that they will take males and females that are closely related, thereby establishing a subcolony in a breeding facility. In such a case, the two colonies will be genetically quite different, and a typical bottleneck effect will occur. Consider a more ideal situation in a rat colony. Suppose that a researcher is working on allotransplantation of a cancer cell line using a closed colony. Assume that the cancer has the A-antigen of RT1. In the closed colony as a recipient of the cancer, the percentages of the animals with A, AB, and B were 25%, 50%, and 25% in one generation. It can be assumed that the cancer will be transplant- able in 75% of the animals with the A-antigen on average. If animals with the 

52 MICROBIAL AND PHENOTYPIC DEFINITION OF RATS AND MICE B-type contributed to the subsequent generation of the divided colony, they will produce only offspring carrying B, which will reject the cancer. Such cases generally do not arise, but the worse case (such as artificially lowering the fre- quency of A-antigen-positive animals) should be carefully considered. DEMONSTRATION OF THE EXISTENCE OF CLOSED COLONIES USING GENETIC MONITORING Closed colonies of rats are known to show genetic polymorphisms at many loci. When they are maintained under unplanned mating, the stocks may show genetic instability. One cause of instability is a bottleneck effect caused by caesarian section, followed by an increase in an inbreeding coefficient in the colony. In this case, the number of loci showing genetic polymorphisms de- creases with each generation. Generally, genetic variation in a closed colony is greater than that seen with an inbred strain. When a colony has been genetically altered for any of the reasons described above, it will be difficult for users of the stocks to interpret their data because genetic changes have occurred. Therefore, we should periodi- cally test closed colonies using genetic methods to confirm genetic stability. From the viewpoint of population genetics, we studied closed colonies of rats for 3 years (1988 to 1990). The Ministry of Education, Science, Culture, and Sports of Japan provided a grant for our study in which we performed genetic analysis on closed colonies of rats with the following aims: (1) to obtain genetic profiles of closed colonies of rats, (2) to obtain monitoring profiles of closed colonies, and (3) to determine specific genes identifying each colony. As men- tioned above, an additional goal of this study was to establish methods of genetic monitoring that are appropriate for closed colonies. Materials and Methods As shown in Figure 4, six closed colonies were obtained from four breeders in Japan: Jcl:Wistar, Crj:Wistar, Iar:Wistar, Jcl:SD, Crj:SD, and Nr:Donryu. Sixty rats consisting of 30 females and 30 males of each colony were used for genetic study. Of the genetic markers used in the study, 21 are biochemical markers and one is an immunological marker (Table 1). Results Gene frequencies and specific markers identifying colony. Gene fre- quencies of 21 biochemical markers are shown in Table 2. Results for RT1 are summarized in Table 3. Markers showing > 0.017 genotypic frequency (equiva- lent to at least one occurrence in 60 animals tested) were counted. Markers identifying colonies were demonstrated as follows: Acon1a: SD stocks; Ahd2b: 

HIDEKI KATOH 53 1920 1930 1940 1950 1960 1970 1980 1990 Wistar Carworth 1970 ? Jcl Farm UK University 1957 Iar 1947 Tokyo Wistar Japan ? CR 1975 CRBL1981 Crj Institute UK USA USA SD hybrid ? hooded 1950 CRBL USA 1964 CR Jcl Wistar 1955 France ? (1925 by R.W.Dawley) Caesarian Section Crj Donryu ? Nr 1920 1930 1940 1950 1960 1970 1980 1990 FIGURE 4. History of the five outbred stocks of the rat used in this study. Jcl:Wistar; Amy1b: Crj:SD; Es3b: Iar:Wistar; Es4c: Nr:Donryu; RT1.Ak: Iar:Wistar; RT1.Ed: Crj:Wistar. Average heterozygosity. As shown in Table 4, percentages of polymorphic loci in six colonies varied from 9.5% (Jcl:Wistar) to 61.9% (SD), and average heterozygosities of six colonies ranged from 0.007 (Donryu) to 0.251 (Crj:SD). Because average heterozygosities of mice and human are 0.078 and 0.099, re- spectively (Nei 1987), those of rat closed colonies were reasonable. Jcl:Wistar revealed the lowest percentage (9.5%) of polymorphic loci. The breeder stated that this colony was reconstituted from several pairs by cesarian section to pro- duce a SPF colony, which might have led to elevation of homozygosity at most loci. Donryu was second in percentage of polymorphic loci and first for average heterozygosity. This ranking was caused by a significant imbalance of allele frequencies of polymorphic loci in the Donryu colony. Genetic distance. In Table 5 are shown the genetic distances among the colonies. Each value represents the level of genetic difference between two colonies. The lowest value was 0.151 between Jcl:SD and Crj:SD, and the highest one was 0.474 between Jcl:SD and Donryu. The close relation between Iar:Wistar and Donryu is shown by a distance value of 0.160. 

54 MICROBIAL AND PHENOTYPIC DEFINITION OF RATS AND MICE TABLE 1 Marker Loci, Samples and Testing Methods Gene Symbol Gene Name Samples Testing Methods Biochemical markers 1 Acon1 Aconitase1 Kidney CAE 2 Ahd2 Aldehyde dehydrogenase 2 Liver IEF (pH3.5-10) 3 Ahdc Aldehyde dehydrogenase c Liver IEF (pH3.5-10) 4 Akp1 Alkaline phosphatase 1 Kidney IEF (pH5-7) 5 Alp1 Serum alkaline phosphatase 1 Kidney IEF (pH5-7) 6 Amy1 Amylase 1 Pancreas CAE 7 Es1 Esterase 1 Plasma CAE 8 Es2 Esterase 2 Plasma PAGE (10% gel) 9 Es3 Esterase 3 Small intestine PAGE (10% gel) 10 Es4 Esterase 4 Kidney CAE 11 Es6 Esterase 6 Testis IEF (pH5-7) 12 Es7 Esterase 7 Testis IEF (pH5-7) 13 Es8 Esterase 8 Testis IEF (pH5-7) 14 Es9 Esterase 9 Testis IEF (pH5-7) 15 Es10 Esterase 10 Testis IEF (pH5-7) 16 Es14 Sex-influenced esterase Plasma CAE 17 Fh1 Fumarate hydratase 1 Kidney CAE 18 Gc Group specific component Plasma PAGE (10% gel) 19 Hbb Hemoglobin beta chain Red blood cells CAE 20 Mup1 Major urinary protein 1 Urine PAGE (15% gel) 21 Svp1 Seminal vesicle protein 1 Seminal vesicle fluid CAE Immunological marker RT1 Histocompatibility 1 Red blood cells Hemagglutination CAE: Cellulose acetate membrane electrophoresis PAGE: Polyacrylamide gel electrophoresis IEF: Isoelectric focusing SUMMARY AND DISCUSSION The results of this study can be summarized as follows: 1. Genes uniquely identifying each colony were found. 2. Lower polymorphism observed in Jcl:Wistar was caused by caesarian section rederivation of the colony. 3. Genetic distance suggested that Crj:SD and Jcl:SD are the closest geneti- cally. It was also revealed that the relation between Iar:Wistar and Donryu was very close yet genetically different from SD and Wistar colonies. Although geneticists and breeders recognize the importance of genetic moni- toring of closed colonies, this issue has not been discussed. The major reason is 

HIDEKI KATOH 55 TABLE 2 Gene Frequencies of 21 Biochemical Markers in Six Outbred Stocks Locus Allele Jcl:Wistar Crj:Wistar Iar:Wistar Jcl:SD Crj:SD Donryu 1 Acon1 a 0.0000 0.0000 0.0000 0.9580* 0.5670* 0.0000 b 1.0000 1.0000 1.0000 0.0420 0.4330 1.0000 2 Ahd2 b 0.1830* 0.0000 0.0000 0.0000 0.0000 0.0000 c 0.8170 1.0000 1.0000 1.0000 1.0000 1.0000 3 Ahdc a 0.0000 1.0000 1.0000 1.0000 0.9020 1.0000 b 1.0000 0.0000 0.0000 0.0000 0.0980 0.0000 4 Akp1 a 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 b 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 5 Alp1 a 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 b 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 6 Amy1 a 1.0000 1.0000 1.0000 1.0000 0.6500 1.0000 b 0.0000 0.0000 0.0000 0.0000 0.3500* 0.0000 7 Es1 a 0.0000 0.0000 0.7000 0.1920 0.3750 1.0000 b 1.0000 0.9670 0.3000 0.0000 0.1670 0.0000 c 0.0000 0.0330 0.0000 0.8080 0.4580 0.0000 8 Es2 a 1.0000 0.9170 0.0000 0.7670 0.3080 0.0000 b 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 c 0.0000 0.0000 0.5420 0.1670 0.0000 0.5920 d 0.0000 0.0830 0.4580 0.0670 0.6920 0.4080 9 Es3 a 0.3330 0.9170 0.0000 0.2920 0.3920 0.9500 b 0.0000 0.0000 0.4250 0.0000 0.0000 0.0000 c 0.0000 0.0000 0.2330 0.4670 0.0000 0.0000 d 0.6670 0.0830 0.3420 0.2420 0.6080 0.0500 10 Es4 a 0.0000 0.0000 0.5420 0.1580 0.0000 0.0000 b 1.0000 1.0000 0.4580 0.8420 1.0000 0.1420 c 0.0000 0.0000 0.0000 0.0000 0.0000 0.8580* 11 Es6 a 1.0000 0.5520 1.0000 0.4330 0.6670 1.0000 b 0.0000 0.4480 0.0000 0.5670 0.3330 0.0000 12 Es7 a 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 b 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 13 Es8 a 0.0000 0.9170 0.4330 0.2330 0.7170 0.1330 b 1.0000 0.0830 0.5670 0.7670 0.2830 0.8670 14 Es9 a 1.0000 0.8330 0.5670 0.7650 0.2830 0.9150 c 0.0000 0.1670 0.4330 0.23350 0.7170 0.0850 15 Es10 a 1.0000 0.9170 0.5670 0.7830 0.2850 0.9170 b 0.0000 0.0830 0.4330 0.2170 0.7150 0.0830 16 Es14 a 1.0000 1.0000 0.7330 0.0000 0.5830 1.0000 b 0.0000 0.0000 0.2670 1.0000 0.4170 0.0000 17 Fh1 a 1.0000 0.0000 0.0000 0.0250 0.0000 1.0000 b 0.0000 1.0000 1.0000 0.9750 1.0000 0.0000 18 Gc a 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 b 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 19 Hbb a 1.0000 0.3170 0.7830 0.4250 0.0000 1.0000 b 0.0000 0.6830 0.2170 0.5750 1.0000 0.0000 20 Mup1 a 0.0000 0.0000 1.0000 0.0000 0.0350 0.9420 b 1.0000 1.0000 0.0000 1.0000 0.9650 0.0580 21 Svp1 a 1.0000 0.5670 1.0000 0.4000 0.7170 1.0000 b 0.0000 0.4330 0.0000 0.6000 0.2830 0.0000 *Bold numbers denote stock-specific antigens. 

56 MICROBIAL AND PHENOTYPIC DEFINITION OF RATS AND MICE TABLE 3 Frequency (%) of Rats Showing Positive Reaction with RT1 Alloantisera Rt1 Antigens Jcl:Wistar Crj:Wistar Iar:Wistar Jcl:SD Crj:SD Donryu Ak 0 0 0.42 0 0 1.00 Au 0.72 0.18 0 0.08 0.72 0 Ed 0 0.65* 0 0 0 0 Aa 0 0.65 0 0.88 0.45 0 Ea 0 0 0 0 0 0 Al 0.75 0.72 0 0.60 0.32 0 ? 0 0 0.58 0.02 0 0 *Bold numbers denotes stock-specific antigen. TABLE 4 Average Heterozygosity of Outbred Stocks of the Rat Outbred Stocks No. of tested loci No. of polymorphic loci H: Average heterozygosity Jcl:Wistar 21 2 (9.5%) 0.0350 Donryu 21 7 (33.3%) 0.0070 Crj:Wistar 21 9 (42.9%) 0.1130 Iar:Wistar 21 9 (42.9%) 0.2030 Jcl:SD 21 13 (61.9%) 0.2020 Crj:SD 21 13 (61.9%) 0.2510 TABLE 5 Genetic Distance Among Six Outbred Stocks of the Rat Jcl:Wistar Crj:Wistar Iar:Wistar Jcl:SD Crj:SD Donryu Jcl:Wistar — 0.237 0.326 0.402 0.414 0.277 Crj:Wistar — 0.261 0.227 0.163 0.336 Iar:Wistar — 0.324 0.232 0.160 Jcl:SD — 0.151 0.474 Crj:SD — 0.446 Donryu — that they have thought for a long time that they could control genetics of the colony through careful breeding schemes. However, as shown in this study, caesarian section produced a bottleneck effect on Jcl:Wistar, and Iar:Wistar is genetically different from Crj:Wistar. Thus, subcolonies exist. The only method for discriminating subcolonies from each other is by genetic testing. For closed colonies, we propose a monitoring method as follows: 

HIDEKI KATOH 57 A. Testing methods The method of the ICLAS Monitoring Center is recommended. We should use the same items for monitoring to facilitate genetic evaluation of each colony. B. Monitoring procedures • Genetic profiling may be performed once at the beginning of periodic monitoring and should be repeated every several years. • Monitoring profiling should be carried out periodically (for example, once a year) using a set of markers selected among the genetic profiling markers shown in Table 2. • Testing numbers should be done, randomly selecting from a production colony with a requirement of 50 to 60 animals per colony. If a breeder has several facilities producing the same stock, all stocks should be tested. REFERENCES Lindsey, J. R. 1979. Historial foundations. In H. J. Baker, J. R. Lindsey, and S. H. Weisbroth, eds. The Laboratory Rat. Vol. 1. Biology and Diseases. Academic Press, New York. Morse, H. C. III. 1981. The laboratory mouse—A historical perspective. In H. L. Fosters, J. D. Small, and J. G. Fox, eds. The Mouse in Biomedical Research. Vol. 1. History, Genetics, and Wild Mice. Academic Press, New York. Nei, N. 1987. Molecular Evolutionary Genetics. Columbia University Press, New York.