The genetic background of laboratory animals can be a critical factor in the success of an experiment. There is no greater testimonial to this statement than our present knowledge of tissue transplantation acquired through the understanding of histocompatibility loci in inbred mice ( 1). One only has to review the historical development of our understanding of mammary carcinoma in mice to appreciate the role of genetically defined animals ( 2).
The National Institutes of Health recently has been designated by the World Health Organization as one of three international genetic resource centers that will provide well-defined and characterized breeding pairs of laboratory animals to any institution involved in biomedical research. The Division of Research Services, which maintains over 100 inbred strains and substrains of mice, therefore, has an obligation to the scientific community to continue the propagation of laboratory animals by the standards of scientific inquiry set forth by the distinguished group of scientists we are honoring at this conference.
The Division of Research Services recently established a genetic monitoring laboratory to ensure that the genetic integrity of the inbred strains and substrains of mice produced by the Veterinary Resources Branch. The genetic monitoring laboratory provides quality assurance through routine monitoring of the inbred colonies of mice for gene definition. The monitoring service provides biochemical and immunologic information for studying and controlling the heredity of laboratory inbred mice. At the present time 33 specific genetic loci located on 13 of the 19 autosomal chromosomes of the mouse are used to define the genetic constitution (genetic profile) of each individual inbred strain and substrain. The genetic markers which constitute the genetic profile fall into three categories: morphological markers, biochemical markers, and immunological markers. Each inbred strain and substrain has a unique distribution of these marker genes which is used to differentiate one strain from another.
Another feature of the genetic monitoring program is the computer-assisted utilization of the genetic profiles in the form of a genetic data bank. The computer can be used to compare several inbred strains of mice for gene similarities and differences, to identify an inbred strain or group of inbred strains by a specific gene distribution, and to aid the research scientist in choosing the appropriate inbred strain for his research project.
Colony Management. The genetic monitoring program is primarily concerned with the genetic quality assurance of the various inbred strains of mice ( Table 1). The program is also involved in all areas of the breeding and maintenance of the colonies which affect the genetic integrity of each inbred strain. Colony management involves the proper execution of the mating system to avoid creating sublines within an inbred strain. In a brother by sister system of mating, the number of generations needed to trace back to a common ancestor should be kept to a minimum. The optimum situation occurs when the breeders at any generation trace back to a common ancestor in two generations. The production demands on a colony regulate the number of breeders which in turn determines the number of generations back to the common ancestor. A second factor that plays a critical role in determining colony size is the reproductive performance of the inbred strain. The number of successful or fertile matings plus litter size influences the breeder count or size of that inbred colony. Good colony management is the first line of defense in protecting the genetic integrity of an inbred strain and is not substituted by sophisticated monitoring or surveillance of the inbred colony.
Training and Consultation. The training of biological laboratory technicians in the principles of simple Mendelian genetics, systems of mating, and genetic monitoring provides a technical staff with an overall understanding of the types of problems that can occur in an inbred animal colony. For example, by knowing the distribution of three coat color genes -- agouti, brown, and albino -- in the inbred strains, the biological laboratory technician can detect mating errors and thus prevent the loss of an entire inbred line. If the biological laboratory technician understands that F1 hybrids from an interstrain cross are all identical, then he can recognize problems of F1-hybrid coat color segregation which could be caused either by a mating error or by a mutation at one of the coat color loci.
The genetic monitoring laboratory can assist the biomedical research scientist to choose the animal strain that best fits the needs of his research program. Attention can be directed to specific genetic markers which are unique to an inbred strain that could influence or interact with the characteristic under investigation.
Genetic Profiles. A genetic profile ( Table 2) of an inbred strain is defined by three types of genetic markers: morphological traits, biochemical characteristics, and immunological markers. The location of the genes controlling these genetic markers on specific chromosomes (chromosome linkage map) and the computerization of this information (genetic data bank) are different forms of a genetic profile and offer different means of using the information.
The concept of a genetic profile is based on the fact that each inbred strain of mice has located on its chromosomes a unique set of genes which distinguishes it from other strains of mice. The development and subsequent maintenance of an inbred strain by a strict system of inbreeding will produce a strain of mice which possesses identical, or nearly identical, sets of pairs of genes. Inbred strains of mice can be grouped into two classes according to their origin. The first category is called inbred strains of independent origin. Inbreeding was started in a group of mice which was not related to and independent of other known colonies of mice. The greatest number of genetic differences would be expected to exist between strains of mice of independent origin. The more widely used strains of independent origin are BALB/c, C57BL, and DBA/2 ( Figure 1).
The second category of inbred mice is called derived inbred strains. Inbred strains in this category were started by first crossing two strains of mice, usually of independent origin, then by inbreeding until genetic characteristics from both parental strains are fixed in the new line. Popular derived inbred strains in use today are A, CBA, and C3H ( Figure 1). Inbred strains which were derived from a non-inbred heterogeneous stock are classified as substrains. Gene similarities and differences between the sublines would depend upon the degree of heterozygosity in the original common stock and upon chance fixation of genes during the inbreeding process. The most widely used groups of inbred strains in this category are the C57BL sublines, C58 and C57BR ( Figure 1).
There are more than 320 mapped genes on the 20 pairs of chromosomes in the laboratory mouse ( 3). For the 19 autosomal pairs of chromosomes our goal is to have at least three genetic markers on each autosome; one locus near each end of the chromosome and one or more loci near the center of the chromosome. At the present time we are using 33 markers on 13 autosomes ( Figure 2 and Table 3). The 33 genetic markers that constitute a genetic profile of an inbred strain have been classified into four functional groups: 1) common morphological characteristics such as coat color and coat texture; 2) non-enzyme proteins found in the serum and red blood cells; 3) enzyme proteins which can be assayed in the liver, blood, kidney, and red blood cell; and 4) immunologic markers such as cell surface antigens of thymocytes and lymphocytes.
These genetic markers represent both qualitative and quantitative gene expression. For example, the isozymes, realized by electrophoretic techniques, of the enzyme dipeptidase ( 4) ( Dip-1, chromosome 1) and kidney esterase ( 5) ( Es-3, chromosome 11) are qualitative expressions of different alleles of structural genes at their respective loci. The electrophoretic isozymes of lactate dehydrogenase regulator ( 6) ( Ldr-1, chromosome 6) and kidney catalase ( 7) ( Ce-2, chromosome 17) represent, respectively, a quantitative expression of a regulator gene, the amount of LDH b-protein present, and a post-translational modification of the enzyme catalase which alters its electrophoretic mobility. Additional examples of quantitative gene expression are erythrocyte catalase ( 8) ( Cs-1, chromosome 2) and deltaaminolevulinate dehydratase ( 9) ( Lv, chromosome 4) which are characterized by conventional spectrophotometric analyses measuring total enzyme activity.
Table 4 is a computerized representation of the classical linkage map ( Figure 2). The loci are listed according to the chromosome on which they are located and are listed in linear order starting from the centromere. The body of the table contains the different alleles carried by each inbred strain. The number of different loci and their order of presentation by the computer can be specified to conveniently compare the genetic profiles of two or more inbred strains. One can look at one locus for a specified inbred strain or look at the entire genotype for all the inbred strains listed in the data bank.
The computer can determine allelic similarities and differences at any number of specified loci between selected inbred strains. Table 5 presents a comparison made by the computer for strains C57BL/10, NBL, and DBA/2. The NBL strain -- prior designation C57BL/10-H-2d or B10B -- was thought to originate as a coisogenic strain with C57BL/10 from a supposed mutation from H-2b to H-2d. Mitchison ( 10) in 1955 and Snell ( 11) in 1958 presented evidence based on tumor transplantation studies that NBL was not coisogenic with C57BL/10. Later in 1963, Finlayson ( 12) showed that the urinary protein (Mup-1, chromosome 4) of NBL was of the same type found in strains BALB/c and DBA/2 -- both H-2d strains -- and different from that found in C57BL/10. These fact prompted the name change from C57BL/10-H-2d to NBL and placed NBL in the category of strains of uncertain origin ( Figure 2). In Table 5 C57BL/10 was specified as the reference strain, and NBL and DBA/2 are compared with C57BL/10 for gene similarities and differences. When a strain has the same allele at a locus as the reference strain, the computer does not print its gene symbol; if the strains differ at a locus, then the computer prints the gene symbol to indicate the difference. For the 19 loci used in the comparison, which represents 11 chromosomes, C57BL/10 and DBA/2 differ by 17 loci, C57BL/10 and NBL differ by 12 loci, and DBA/2 and NBL differ by six loci. Although BALB/c and NBL only differ by seven of the 19 loci used for comparison, BALB/c can be eliminated as a possible outcross strain on the basis of four loci. NBL and DBA/2 have the same alleles at Id-1 and Dip-1 (chromosome 1), Hc (chromosome 2), and the Ly-2,3 complex (chromosome 6), whereas BALB/c and C57BL/10 have the alternate genes at these loci. The strain comparison supports the conclusions from the transplantation studies that NBL was the result of a C57BL/10 outcross and also indicates that DBA/2 is the most likely candidate as the outcross strain.
Another way to utilize the genetic data bank is to have the computer search the strain profiles for inbred strains with a specific gene or set of genes. In this way, possible linkage associations can be detected for an unlinked gene with a gene located on a specific chromosome. For example, after classifying the inbred strains for kidney catalase (Ce-2) and entering the data into the data bank, a search was made for the strains carrying the allele Ce-2b. A computer listing ( Table 6) indicated that seven inbred strains were classified as Ce-2b. Strain profiles for the seven strains showed that H-2kk (chromosome 17) was present in all seven strains. A search for the strains carrying H-2Kk listed nine inbred strains with this allele ( Table 7). Seven of the nine strains are Ce-2b, suggesting that Ce-2 is located on chromosome 17. A linkage analysis of the first backcross mice confirmed the linkage of Ce-2 and H-2K and placed Ce-2 two map units from H-2K.
It is noteworthy that in light of this close linkage that the exceptions to the Ce-2b and H-2Kk association are C58 and C57BR. These two strains have a common origin ( Figure 1) and are genetically independent of the other H-2k inbred strains. This could indicate a possible subtle difference between H-2k haplotypes from different gene pools.
Genetic Surveillance. Progeny from foundation colony breeders which contribute to the main line of descent of an inbred strain are monitored for their conformity with their strain profile. Four to ten chromosomes are routinely examined every generation, and every third generation a complete genetic profile is performed on each inbred strain. Tissues from the breeding pairs destined to become the common ancestors are frozen and stored in liquid nitrogen. In this way genetic profiles from mice of the current generation of inbreeding can be compared with their ancestors several generations removed.
Considerable time and effort have been invested in the genetic monitoring program because we believe that these profiles will vastly improve the validity and reproducibility of research investigations. Not only can we control the genetic integrity of the inbred strains of mice through our genetic profile system, but we can selectively breed mice that have a specific set of biochemical and immunologic characteristics. The genetic profiles have been computerized for ready availability and we are encouraging the development of new animal models. For specific research projects, anyone desiring to attempt such a program is encouraged to consult with the NIH genetic monitoring laboratory for collaborative work along these lines.
1. Klein, J. (1975). Biology of the Mouse Histocompatibility-2 Complex. Springer-Verlag, New York.
See also MGI.
2. Heston, W. (1974). J. Hered. 65: 262.
See also PubMed.
3. Mouse News Letter. (1977). 57: 6.
4. Lewis, W., and Truslove, G. (1969). Biochem. Genet. 3: 493.
See also MGI.
5. Ruddle, F., and Roderick, T. (1965). Genetics 51: 445.
6. Hutton, J., and Roderick, T. (1970). Biochem. Genet. 4: 339.
See also MGI.
7. Hoffman, H., and Grieshaber, C. (1976). Biochem. Genet. 14: 59.
See also MGI.
8. Feinstein, R., Howard, J., Braun, J., and Seaholm, J. (1967). Genetics 56: 559.
9. Russell, R., and Coleman, D. (1963). Genetics 48: 1033.
See also MGI.
10. Mitchison, N. (1955). J. Exp. Med. 102: 157.
11. Snell, G. (1958). J. Natl. Cancer Inst. 21: 843.
See also MGI.
12. Finlayson, J., Potter, M., and Runner, C.R. (1963). J. Natl. Cancer Inst. 31: 91.
See also MGI.