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Genetic and Phenotypic Definition of Laboratory Mice and Rats / What Constitutes an Acceptable Genetic-Phenotypic Definition Muriel T. Davisson Senior Staff Scientist, The Jackson Laboratory Bar Harbor, Maine GENETICALLY DEFINED MICE Although âgenetically definedâ is often equated with inbred strains, a geneti- cally defined strain is any strain in which the genetic background is known, is similar or identical from one mouse to another, and can be faithfully reproduced over time. Genetically defined mice are important for basic and biomedical research. They provide reproducible systems that enable investigators to repli- cate experiments and enable different scientists to use genetically similar or identical research animal models. This presentation discusses key elements in the use of genetically defined mice: genetic standardization, standard genetic nomenclature, genetic definitions of different types of strains, and the value and uses of different types of genetically defined strains. The focus of this presenta- tion is the laboratory mouse, Mus. Mouse models are surrogates for human conditions, but they need not precisely replicate a human disease to be of value for biomedical research. More importantly, a model should be genetically defined so that the results observed can be attributed to the gene or genes being studied and the experiments can be replicated. GENETIC STANDARDIZATION Genetic standardization means simply that a related group of individuals can be genetically described, are similar to each other, and can be recreated by a standard and defined breeding protocol. The value of genetically standardized 63
64 MICROBIAL AND PHENOTYPIC DEFINITION OF RATS AND MICE models is that they can be repeatedly reproduced simply by breeding. A model system is of little value unless it can be propagated reliably. Such a model assures the continued availability of the same model to differ- ent investigators at different institutions over long periods of time. Experiments can be replicated for verification of data and experiments in one laboratory or repeated in another with the expectation that results will be similar if they are due to the mutant gene or genes being studied. It is critical to state in publications the genetic background of the mice studied so other scientists can repeat your experi- ment. One of the reasons for concern about genetic definition is that with the recent strong emphasis on training in molecular biology, scientists often have an inad- equate understanding of whole animal biology, classical genetics, breeding mice, maintaining strains, and keeping pedigree records or a lack of appreciation for why it makes a difference. Much of the literature published on targeted mutation mice and transgenic mice is compromised by lack of a clear definition of the genetic background on which the mutation was studied. It is well known among mouse and human geneticists that genetic heteroge- neity can alter the phenotypic expression of identical mutant genes in different individuals. This phenomenon is thought to contribute to much of the variability among human beings with the same genetic disease. Thus, it is important to keep the genetic background as homogeneous as possible when trying to determine the effects of a mutated gene in a model system. Inbred laboratory mice provide the possibility to do this. Individual mice within an inbred mutant strain are essen- tially genetically identical to each other except for the mutant gene being studied. Differences between mutant and nonmutant (control) mice can be attributed to the mutant gene with a high degree of certainty. Different mouse strains are known to have different behavioral and pheno- typic characteristics. Different strain backgrounds can alter the phenotypic ef- fects of individual major genes. There are many examples of spontaneous or targeted mutations producing different phenotypes when they are transferred from one genetic background to another. If the strain used is not genetically defined, one cannot really know what aspects of the phenotype being studied are due to genetic background effects or to the mutation itself. GENETIC NOMENCLATURE Standard genetic nomenclature provides unique identification for different strains. Investigators reading a paper can obtain the appropriate animals to repli- cate the experiments described or carry out related experiments in the same system. The strain symbol also conveys basic information about the type of strain or stock used and the genetic content of that strain. Examples of symbols for different types of strains are given in the next section, describing the values of different types of genetically defined strains. Rules for symbolizing strains and
MURIEL T. DAVISSON 65 stocks have been promulgated by the International Committee on Standardized Genetic Nomenclature for Mice since the early 1950s. The rules are available on- line from the Mouse Genome Database (MGD; http://www.informatics.jax.org) and were most recently published in print copy (Lyon and others 1996). Strain symbols typically include a Laboratory Registration Code (Lab Code). The first Lab Code appended to a strain symbol identifies and credits the creator of the strain. The Lab Code at the end of a strain symbol indicates the current source for obtaining mice of that strain. Different Lab Codes appended to the same strain symbol distinguish sublines and alert the user that there may be genetic diver- gence between the different sublines. For example, CBA/J is known to have genetic differences from CBA/CaJ. Lab Codes are assigned from a central regis- try to assure that each is unique. The registry is maintained at the Institute for Laboratory Animal Research (ILAR) at the National Academy of Sciences, Wash- ington, D.C. Lab Codes may be obtained electronically at ILARâs web site (www4.nas.edu/cls/ilarhome.nsf). DEFINITION AND VALUE OF DIFFERENT KINDS OF STRAINS Inbred strains are defined as having been created by more than 20 genera- tions of sibling or filial matings (symbolized F20). In reality, a strain will not be completely homozygous at all loci until it has been propagated for more than 40 generations because residual heterozygosity still can be detected at 40 genera- tions (Fox and Witham 1997). Individuals of an inbred strain are considered to be genetically identical, and phenotypic variations are due to environmental dif- ferences. Because individuals are genetically identical, studies can be done with relatively small sample sizes. Inbred strains are valuable to define the genetics of traits such as susceptibility to infectious disease or response to specific drugs. Inbred strains that differ in such traits can be crossed together to define the genetic basis of the differences and to determine the number and chromosomal location of genes involved. They also are used when multiple genetically identi- cal animals are needed to test the effects of a treatment. Inbred strains are typically symbolized by a few capitalized Arabic letters followed by a forward slash and a subline number and/or Lab Code. For example, CBA/CaJ is the subline of CBA inbred strain maintained first by Carter (Ca) and now by The Jackson Laboratory (J). One should be aware that conclusions drawn from studying a single inbred strain apply only to that inbred strain. For example, if you study response to treatment with some agent, your results are specific only to that particular inbred strain. You cannot generalize across many inbred strains. If you want to model a noninbred human population, you might want to use an outbred mouse popula- tion of some kind and look for variability in response to your treatment. Because some variation will be due to genetic variability, larger sample sizes are required than for experiments with inbred mice. A caveat about outbred mice, however, is
66 MICROBIAL AND PHENOTYPIC DEFINITION OF RATS AND MICE that mice coming from small closed colonies are often not as outbred as we think they are. Also, a stock that has been rederived to improve its health status has been through a breeding bottleneck that will reduce the heterogeneity in the stock. Hybrid mice are made by crossing mice of two inbred strains together. The resulting F1 hybrids are genetically identical because at each gene they all carry both alleles from the two inbred parents. Their uses are similar to inbred strains but they are more robust. F1 hybrids cannot be self-propagated and must be created each time by mating mice from two inbred strains. Hybrid mice are symbolized using abbreviations for the parental strains. Their symbols, when correctly written, indicate the sex of each parent. For example, a B6D2 F1 hybrid is created by mating a C57BL/6 (B6) female to a DBA/2 (D2) male; a D2B6 F1 is created by mating a DBA/2J female to a C57BL/6J male. Inbred mutant strains are inbred strains that carry one or more spontaneous or induced single gene mutations. Such strains differ from the parental strain only by the mutated gene (and in some cases closely linked genes; see below). They are valuable for understanding the effects of single gene mutations and for cloning disease genes. Differences in phenotype between mutant mice and con- trol littermates or same strain control mice can be attributed to the mutated gene. There are two kinds of inbred mutant strains. Coisogenic mutant strains are the original strains on which the mutations occurred, and mutant mice differ from control mice only by the mutant gene. Congenic mutant strains carry a mutation that has been backcrossed onto the strain background from another strain or noninbred stock background. The nonrecombinant DNA around the mutation is from the original donor strain. This distinction is important when positionally cloning genes because in the congenic strain, differences in any genes considered candidates for the mutation may be polymorphic differences transferred with the mutation from the original strain. Mixed inbred strains are inbred strains that are recently derived from two inbred genomes. A common example would be when targeted mutation strains are derived by sibling matings starting with the chimeric founder, composed of cells from the 129 embryonic stem cells and the host, typically C57BL/6J, mated to a littermate. Such a strain is designated using the abbreviations for the two parental âstrainsâ separated by a comma, such as B6, 129. In a segregating inbred strain, the mutation is maintained with forced hetero- zygosity by intercrossing heterozygotes or mating heterozygotes Ã homozygotes. In either case, both mutant and control animals are present within the same strain. In homozygous mutant strains, wild-type mice of the same background strain must be used as controls. Recombinant inbred (RI) strains are sets of inbred strains created from sibmated F2 progeny produced by crossing mice from different inbred progenitor strains, such as C57BL/6J and DBA/2J. RI strains are valuable for mapping phenotypic or quantitative traits that differ between the progenitor strains. They
MURIEL T. DAVISSON 67 are especially valuable for controlling for environmental variability in a trait because several genetically identical mice from each line in a set can be typed to score the line for a trait. Crossover events can be detected by strain distribution patterns (SDPs) of alleles among the RI lines, typically using a series of regional markers (Bailey 1971; Taylor 1989; Mouse Genome Database) (Table 1). RI strain sets are like a linkage cross-frozen in time, and genotyping is cumulative. Recombinant congenic strains are sets of inbred strains derived in a similar manner to RI sets except that one or more backcrosses to one parental (designated the background) strain are made after the F1 generation, before inbreeding is begun. The other parental strain is designated the donor. The proportion of background and donor genomes is determined by the number of backcrosses preceding inbreeding. Because these sets are typically constructed after two backcrosses, each recombinant congenic strain usually contains approximately 87.5 % of its genes from the background strain and approximately 12.5% of its genes from the donor strain (Moen and others 1991; Stassen and others 1996). As with recombinant inbred strains, a detailed characterization of SDPs of genes within a strain set may be used to determine linkage relationships between loci and chromosomal segments associated with a trait such as tumor susceptibility. Typing of recombinant congenic strains is useful in the analysis of complex genetic traits in the mouse (Moen and others 1991). Congenic strains are derived by successive backcrosses in which one strain (the donor) donates a segment of chromosome to the recipient (background or host) strain. Congenic strains are genetically almost identical to the background strain except for a short chromosomal segment contributed by the donor strain. The most familiar congenic strains are histocompatibility congenics (Snell and Bunker 1965). More detailed information on strains of laboratory mice may be found in The Jackson Laboratoryâs Handbook on Genetically Standardized JAX Mice (1997). GENETIC MONITORING Genetic monitoring is critical to maintaining genetically defined strains. Although this topic is covered in another presentation, I touch briefly on it here. The best protection against genetic contamination is good animal husbandry and record keeping. There is no substitute. Genetic monitoring is just what its name describesâmonitoring to assure that mistakes have not been made. There are two kinds of genetic monitoring: (1) genetic background monitoring to detect and eliminate possible genetic contamination, and (2) mutation monitoring to assure that the mutation carried by a mutant strain is still present. Neither is particularly difficult to do, but both are crucial to ensure strain integrity and, in the case of mutant strains, avoid the loss of valuable mutations. Genetic background moni- toring is typically done by screening a set of biochemical and DNA markers in progenitor breeding pairs, that is, the breeding pairs in each, or at least every
68 MICROBIAL AND PHENOTYPIC DEFINITION OF RATS AND MICE other, generation that are in the straight line pedigree for the strain. It also is wise, especially in large colonies with several generations of expansion, to moni- tor mice chosen randomly from the expansion colony. Because this type of monitoring is retrospective, a change might not be detected until it is widespread. One needs only a minimum set of markers whose allele distribution distinguishes that strain from others in the same mouse room. Coat color is a simple visible marker that requires no genotyping. Its use can be enhanced by simply inter- spersing strains of different coat colors in the same mouse room. In addition, genetic contamination between different strains usually results in a sudden in- crease in reproductive performance. Be suspicious if mice from a strain with low reproductive performance suddenly start to breed well. Monitoring spontaneous mutations can be as simple as visually observing mice in each generation for the mutant phenotype. Although this is usually adequate, if the phenotype is common to multiple nonallelic mutations, there is a risk that one mutation may be lost and replaced by another in a segregating mutant strain. For example, all mutations that affect the cerebellum cause very similar balance defects. Once a spontaneous mutation is cloned, it can be followed in nonaffected carriers or verified periodically by DNA genotyping. Targeted and induced mutations and transgenes also can be monitored visually if there is an associated phenotype. For those in which the mutants die during gestation, DNA genotyping may be used to follow the mutated gene or transgene. If several targeted mutations have been created using the same type of construct, the same polymerase chain reaction (PCR) protocol can be used to economically genotype mice of all strains. For example, a set of targeted mutation strains made with neo- containing targeting vectors can be typed simultaneously PCR-typing for neo. However, one should periodically genotype each strain with allele-specific markers to ensure against cross-contamination between such strains. GENETIC DATABASES Several databases are available that have information on genetically stan- dardized mice. The Mouse Genome Database mentioned above has a list of inbred strains of laboratory mice, as well as information on mouse genomics and gene expression. The strain list is prepared by Dr. Michael Festing of England, who also is responsible for assigning strain names to new inbred strains. The Laboratory Registration Code database is maintained at ILAR, also mentioned above. The Jackson Laboratory web site (http://www.jax.org) has two databases that list targeted mutation and transgenic mice: (1) TBASE, the transgenic data- base, which was developed by Dr. Rick Woychik, was transferred in 1998 from The Johns Hopkins University; and (2) the Induced Mutant Resource (IMR) database, which lists induced mutant strains available from The Jackson Labora- tory. Both are supported by the National Center for Research Resources (NCRR).
MURIEL T. DAVISSON 69 Scientists using mice also should know how to find other speciesâ databases because it is important to try to give homologous genes in different species the same or similar symbols. Some databases for species most commonly referred to in comparative studies are listed in Table 1. TABLE 1 Selected Genetic and Strain Databases Available on the World Wide Web (WWW) Site Contents Web address (URL) Mouse Genome Database Mapping data (all techniques) http://www.informatics.jax.org genetic, cytogenetic, physical, and comparative mapping data MRCa Mammalian Comparative maps, strain list http://www.mgu.har.mrc.ac.uk/ Genetics Unit The Whole Mouse Catalog Links to web sites for mouse http://www.rodentia.com/wmc (formerly Mice and Rats and rat research Home Page) Animal Genome Database Mouse genetic mapping data, http://ws4.niai.affrc.go.jp/ in Japan cytogenetic maps Human Genome Databaseb Human gene symbols http://bioinfo.sickKids.on.ca/ http://gdbwww.gdb.org/ Human Gene Nomenclature Human gene symbols http://www.gene.ucl.ac.uk/ Database cgi-bin/nomenclature/ searchgenes.pl National Center for Mouse/human comparative http://www.ncbi.nlm.nih.gov/ Biotechnology maps, links to other Homology/ Information (NCBI) databases Rat Genome Database Rat genetics http://ratmap.gen.gu.se/ Roslin Institute Pig, sheep, cattle, chicken http://www.ri.bbsrc.ac.uk/ Bioinformatics bioinformatics/ FlyBase Drosophila genomics http://flybase.bio.indiana.edu http://shigen.lab.nig.ac.jp:7081 Zebrafish Informatics Zebrafish genomics http://zfish.uoregon.edu/ZFIN/ aMRC, Medical Research Council. bNote: At the time of this writing, the Human Genome Database is in transition between the two sites listed.
70 MICROBIAL AND PHENOTYPIC DEFINITION OF RATS AND MICE TRAINING SCIENTISTS TO USE GENETICALLY DEFINED MICE Finally, I would like to return to the point that many scientists trained in the 1980s and 1990s have not really been trained in practical genetics or animal husbandry. With the current research trend moving back toward phenotype analysis, mutagenesis, and whole animal studies, there is a desperate need to provide programs that train scientists to understand, work with, and maintain genetically defined mice. We need resources to provide training in practical genetics, breeding schemes, record keeping, and mouse husbandry. I think an increasing number of investigators today recognize the effects of genetic back- ground on phenotype and the importance of using genetically defined strains; however, many need resources to help them with the practical aspects of creating and using such strains. For example, The Jackson Laboratory has an annual course called Experimental Genetics that is geared to graduate students, post- doctoral fellows, and investigators changing their research programs to use mice. The course teaches practical Mendelian genetics, how to breed animals, how to keep records to avoid mixing up mice within the colony, and basic animal hus- bandry. Unfortunately, the course handles only about 30 students a year and, as far as I know, is the only course of its type in this country. We need more of this sort of course introduced into graduate schools or offered in training programs similar to that at The Jackson Laboratory. REFERENCES Bailey, D. W. 1971. Recombinant-inbred strains. An aid to finding identity, linkage, and function of histocompatibility and other genes. Transplantation 11:325-327. Fox, R. R., and B.Witham, editors. 1997. Handbook on Genetically Standardized JAX Mice. The Jackson Laboratory, Bar Harbor, Maine. Lyon, M. F., S. Rastan, and S.D.M. Brown. 1996. Genetic Variants and Strains of the Laboratory Mouse. Oxford University Press, Oxford. Moen, C. J., M. A.van der Valk, M. Snoek, B. F. van Zutphen, O. von Deimling, A. A. Hart, and P. Demant. 1991. The recombinant congenic strainsâA novel genetic tool applied to the study of colon tumor development in the mouse. Mamm. Genome 1:217-227. Snell, G. D., and H. P. Bunker. 1965. Histocompatibility genes of mice. V. Five new histocompat- ibility loci identified by congenic resistant lines on a C57BL/10 background. Transplantation 3:235-252. Stassen, A. P., P. C. Groot, J. T. Eppig, and P. Demant. 1996. Genetic composition of the recombi- nant congenic strains. Mamm. Genome 7:55-58. Taylor, B. A. 1989. Recombinant inbred strains. Pp. 773-796 in M. F. Lyon and A. G. Searle, eds. Genetic Variants and Strains of the Laboratory Mouse. 2nd edition. Oxford University Press, New York.