Inbred Strains of Mice

The Generation of Inbred Strains • The Classical Inbred Strains • Segregating Inbred Strains • Newly Derived Inbred Strains

The offspring that result from a mating between two F1 siblings are referred to as members of the "second filial generation " or F2 animals, and a mating between two F2 siblings will produce F3 animals, and so on. An important point to remember is that the filial (F) generation designation is only valid in those cases where a protocol of brother-sister matings has been strictly adhered to at each generation subsequent to the initial outcross. Although all F1 offspring generated from an outcross between the same pair of inbred strains will be identical to each other, this does not hold true in the F2 generation which results from an intercross where three different genotypes are possible at every locus. However, at each subsequent filial generation, genetic homogeneity among siblings is slowly recovered in a process referred to as inbreeding. Eventually, this process will lead to the production of inbred mice that are genetically homogeneous and homozygous at all loci. The International Committee on Standardised Nomenclature for Mice has ruled that a strain of mice can be considered "inbred" at generation F20 (Committee for Standardised Genetic Nomenclature for Mice, 1989).

The process of inbreeding becomes understandable when one realises that at each generation beyond F1, there is a finite probability that the two siblings chosen to produce the subsequent generation will be homozygous for the same allele at any particular locus in the genome. If, for example, the original outcross was set up between animals with genotypes A/A and a/a at the A locus, then at the F2 generation, there would be animals with three genotypes A/A, A/a, and a/a present at a ratio of 0.25:0.50:0.25. When two F2 siblings are chosen randomly to become the parents for the next generation, there is a defined probability that these two animals will be identically homozygous at this locus. Since the genotypes of the two randomly chosen animals are independent events, one can derive the probability of both events occurring simultaneously by multiplying the individual probabilities together according to the "law of the product". Since the probability that one animal will be A/A is 0.25, the probability that both animals will be A/A is 0.25 x 0.25 = 0.0625. Similarly, the probability that both animals will be a/a is also 0.0625. The probability that either of these two mutually exclusive events will occur is derived by simply adding the individual probabilities together according to the "law of the sum" to obtain 0.0625 + 0.0625 = 0.125.

If there is a 12.5% chance that both F2 progenitors are identically homozygous at any one locus, then approximately 12.5% of all loci in the genome will fall into this state at random. The consequence for these loci is dramatic: all offspring in the following F3 generation, and all offspring in all subsequent filial generations will also be homozygous for the same alleles at these particular loci. Another way of looking at this process is to consider the fact that once a starting allele at any locus has been lost from a strain of mice, it can never come back, so long as only brother-sister matings are performed to maintain the strain.

At each filial generation subsequent to F3, the class of loci fixed for one parental allele will continue to expand beyond 12.5%. This is because all fixed loci will remain unchanged through the process of incrossing, while all unfixed loci will have a certain chance of reaching fixation at each generation. At each locus which has not been fixed, matings can be viewed as backcrosses, outcrosses, or intercrosses, which are all inherently unstable since they can all yield offspring with heterozygous genotypes.

Figure 3.2 shows the level of homozygosity reached by individual mice at each generation of inbreeding along with the percentage of the genome that is fixed identically in both animals chosen to produce the next filial generation according to the formulas given by Green, 1981. After 20 generations of inbreeding, 98.7% of the loci in the genome of each animal should be homozygous (Green, 1981). This is the operational definition of inbred. At each subsequent generation, the level of heterozygosity will fall off by 19.1%, so that at 30 generations, 99.8% of the genome will be homozygous and at 40 generations, 99.98% will be homozygous.

These calculations are based on the simplifying assumption of a genome that is infinitely divisible with all loci assorting independently. In reality, the size of the genome is finite and, more importantly, linked loci do not assort independently. Instead, large chromosomal chunks are inherited as units, although the boundaries of each chunk will vary in a random fashion from one generation to the next. As a consequence, there is an ever-increasing chance of complete homozygosity as mice pass from the 30th to 60th generation of inbreeding (Bailey, 1978). In fact, by 60 generations, one would be virtually assured of a homogeneous homozygous genome if it were not for the continual appearance of new spontaneous mutations (most of which will have no visible effect on phenotype). However, every new mutation that occurs will soon be fixed or eliminated from the strain through further rounds of inbreeding. Thus, for all practical purposes, mice at the F60 generation or higher can be considered 100% homozygous and genetically indistinguishable from all siblings and close relatives (Bailey, 1978). All of the classical inbred strains have been inbred for at least 60 generations.

The Classical Inbred Strains
During the first three decades of the twentieth century, a series of inbred strains were developed from mice obtained through the fancy trade. A small number of these "classical strains" have, through the years, become the standards for research in most areas of mouse biology. The most important of these strains are listed in Table 3.1 along with their uses, other characteristics, and the number of generations of sequential brother-sister matings that had been accomplished, as of 1993, in the colonies of the major suppliers. Other characteristics relevant to the reproductive performance of many of the classical inbred strains are tabulated in Table 4.1. Pictures of several classical and newly derived mouse strains are presented in Figure 3.3.

Segregating Inbred Strains
A special class of inbred strains are produced and maintained by brother-sister mating in the same manner just described with one major exception. Instead of selecting animals randomly at each generation for further matings to maintain the strain, an investigator purposefully selects individuals heterozygous for a mutant allele at a particular locus of interest. This "forced heterozygosity" at each generation results in the development of a "segregating inbred strain" with the same properties as all other inbred strains in regions of the genome not linked to the "segregating locus". In almost all cases, segregating inbred strains are developed around mutant loci that cause lethality, severely reduced viability, or sterility in the homozygous state. Some mutant genes - including Steel (Sl), Yellow (Ay), Brachyury (T) and Disorganisation (Ds) - can be recognized through the expression of a dominant phenotype that allows direct selection of heterozygotes at each generation. With other mutant genes, heterozygotes cannot be recognized directly and must be identified by progeny testing or through closely linked marker alleles that are recognizable in the heterozygous state.

At each generation of breeding, a segregating inbred strain will produce two classes of animals: those that carry the mutant allele and those that do not. Thus, it is possible to use sibling animals as "experimental" and "control" groups to investigate the phenotypic effects of the mutation in a relatively uniform genetic background. Segregating inbred strains are conceptually similar to congenic strains.

Newly Derived Inbred Strains
When the genomes of the traditional inbred strains were first analysed with molecular probes during the 1980s, it became clear that their common origin from the fancy mouse trade had led to a great reduction in inter-strain polymorphism at many loci. Since polymorphisms are essential for formal linkage analysis, crosses between the traditional inbred strains were less than ideal for this purpose. This problem could be overcome with the development of new inbred strains that were genetically distinct from the traditional ones. Another driving force in the development of new strains from scratch was the realization that none of the traditional strains were derived from a single subspecies or population; instead, they were all undefined genomic mixtures from two or more subspecies. Thus, the classical laboratory mice do not actually represent any animal that exists in nature. Although for many investigators, this would not appear to be an important problem, it is likely to become more relevant in future studies that are focused on the interactions among multiple genes rather than single genes in isolation. Within the traditional strains, unnatural combinations of alleles could have subtle unnatural effects on the operation of polygenic traits. To overcome this problem, new inbred strains are routinely derived from a pair of animals captured from a single well-defined wild population. Over the last several decades, inbred strains have been developed from animals representing each of the major subspecies in the house mouse group as well as somewhat more distant species that still form fertile hybrid females with M. musculus.

The major hurdle that must be overcome in the development of new inbred strains from wild populations is inbreeding depression, which occurs most strongly between the F2 and F8 generations. The cause of this depression is the load of deleterious recessive alleles that are present in the genomes of wild mice as well as all other animal species. These deleterious alleles are constantly generated at a low rate by spontaneous mutation but their number is normally held in check by the force of negative selection acting upon homozygotes. With constant replenishment and constant elimination, the load of deleterious alleles present in any individual mammal reaches an equilibrium level of approximately ten. Different unrelated individuals are unlikely to carry the same mutations, and as a consequence, the effects of these mutations are almost never observed in large randomly-mating populations.

Thus, it not surprising that during the early stages of mouse inbreeding, many of the animals will be sickly or infertile. At the F2 to F8 generations, the proportion of sterile mice is often so great that the earliest mouse geneticists thought that inbreeding was a theoretical impossibility (Strong, 1978). Obviously they were wrong. But, to succeed, one must begin the production of a new strain with a very large number of independent F1 X F1 lines followed by multiple branches at each following generation. Most of these lines will fail to breed in a productive manner. However, an investigator can continue to breed the few most productive lines at each generation - these are likely to have segregated away most of the deleterious alleles. The depression in breeding will begin to fade away by the F8 generation with the elimination of all of the deleterious alleles. Inbreeding depression will not occur when a new inbred strain is begun with two parents who are themselves already inbred because no deleterious genes are present at the outset in this special case.

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