Inbred Strains of Mice
The Generation of 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
Segregating Inbred Strains
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
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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