A 129

a C57BL was separated into two major substrains.

Inbred strains are also of increasing importance as the "genetic background" for many mutants, "knockouts," and transgenic alterations. This is discussed in more detail below (see congenic strains). At least 17 Nobel prizes have been awarded for work that required inbred strains or would have been much more difficult without them.35

There are over 400 "straight" inbred strains of mice and about 200 inbred strains of rats, excluding congenic, recombinant, etc., strains discussed below, each of which is an inbred strain in its own right. The origin and phenotypic characteristics of these strains is available on the Jackson Laboratory and Rat Genome Database Web sites. Extensive genealogies of inbred mouse strains have also been developed.36 However, probably 80% of research using inbred strains is done using about ten to 15 of the most common strains. The estimated "Top Ten" strains of mice and rats, according to the known number of laboratories maintaining each strain,37 are listed in Table 9.5

There are two main reasons for choosing a strain such as C57BL/6 mice or F344 rats for a particular research project. The strain may be chosen because it is regarded as a good general-purpose strain, which can be used to replace the use of an outbred stock such as Swiss mice or Wistar rats. In this case, a strain would be chosen that has no known characteristics which would preclude its use for the project. Strains with a high incidence of a specific disease, which were highly aggressive, or which had some known immunological defects, for example, might not be suitable for the project. When choosing a suitable strain for a new project, several available strains could be screened to find one that showed an appropriate response to the treatment of interest. The project could then be continued with that strain, with occasional studies of other strains just to confirm that the results were not highly strain specific.

In contrast, a specific strain may be chosen because it has characteristics that make it useful for a particular type of research. For example, C57BL/6 mice maintained on a high-fat diet develop atherosclerosis, so they may be used as a model of that condition in humans.36 C57BL/6 mice also like sweet tastes and alcohol and are highly active in an open field. A searchable database of these sorts of phenotypic characteristics of mouse and rat strains is available on the Web. Many strains are used for studying QTLs associated with phenotypes ranging from susceptibility to cancer to various aspects of behavior, growth, metabolic diseases, and immune function. A database of genetic markers in 55 mouse strains (http://www.cidr.jhmi.edu/mouse/mouse.html) developed at the Center for Inherited Disease Research makes it extremely easy to pick informative genetic markers in proposed crosses between any two strains.

Breeding and Maintenance of Inbred Strains

The usual aim in maintaining a colony of laboratory animals is to prevent genetic change so that information on the characteristics of the strain can be accumulated and used in the planning and interpretation of future experiments. Provided genetic contamination due to a mating with an animal of another strain can be ruled out, the only way in which inbred strains can change is as a result of the accumulation of new mutations or from residual heterozygosity; the segregation polymorphisms still remaining in the strain after 20 generations of inbreeding. An economical and flexible breeding program that can rapidly respond to changes in demand is also wanted.

Figure 9.2 Diagram of the "traffic light" system for controlling the foundation stock (FS) and multiplication colonies of an inbred strain. The FS colony is self-perpetuating, and any surplus stock can be transferred to the multiplication colony in breeding cages with a white label. Their offspring can be used for a further three generations of multiplication, but the offspring of the red label colony are not used for breeding.

Figure 9.2 Diagram of the "traffic light" system for controlling the foundation stock (FS) and multiplication colonies of an inbred strain. The FS colony is self-perpetuating, and any surplus stock can be transferred to the multiplication colony in breeding cages with a white label. Their offspring can be used for a further three generations of multiplication, but the offspring of the red label colony are not used for breeding.

Unlike outbred stocks, inbred strains can be maintained with very small numbers of breeding stock. Inbred mice and rats are usually maintained as permanently mated monogamous pairs, and the minimum-sized breeding colony is one that just prevents the colony from dying out, given that breeding performance is often somewhat uncertain, particularly with some strains. Where large numbers of animals are needed for research purposes, an appropriate breeding scheme is to maintain a "stem-line" colony of about ten to 30 breeding pairs, with a multiplication colony of sufficient size to provide all the required experimental animals as a result of up to about four generations of breeding. The multiplication colony is used only to produce experimental animals and does not contribute to the long-term survival of the strain. This breeding scheme is shown diagrammatically in Figure 9.2.

The stem-line colony should be maintained by brother x sister mating, usually as pairs, and should, as far as possible, be kept physically separated from all other animals of the same species. Some breeders maintain such colonies in isolators or as frozen embryos. Genetic quality control methods (see below) should be used to authenticate the strain and monitor it over a period of time. Detailed records on each breeding pair should be maintained, as well as a pedigree chart showing the relationship between the pairs. These can be paper or computer-based records. Generally, all breeding pairs should be descended from a single breeding pair about five to seven generations back. Although directional selection within an inbred strain should have no effect, it is advisable to select for good breeding performance in order to try to prevent it from declining as a result of the accumulation of new deleterious mutations. Often, such mutations will have a very small effect, and the best method of selection is probably to base it on the average performance of the various sublines in the colony. For example, an index of productivity of each breeding pair, such as the number of young weaned per pair per week, can be recorded on the pedigree chart. Replacement breeding stock would then be chosen according to the average productivity of the different substrains, though a substantial rise in productivity could indicate genetic contamination with consequent hybrid vigor. A simplified example is given in Figure 9.3.

The multiplication colony should also be physically separated from other colonies of the same species as far as possible, in order to prevent genetic contamination with a nonstrain mating. The colony may be sib-mated if the aim is to identify any new mutations that may occur, as this will tend to reveal any recessive mutations. Alternatively, if the aim is simply to produce large numbers of experimental animals, the colony can be random mated, and trios or other mating systems can be used if these are more economical. Random mating for a few generations should have no adverse impact on the genetic quality of the animals. A few new mutations may accumulate, but in practice, these are unlikely to have any impact on research. However, as noted above, a maximum of about four generations of such mating should be used. A practical scheme for the multiplication colony is to use the "traffic light" scheme,38 in which surplus offspring of the stem-line go into breeding cages with a white label, their offspring go into cages with a green label, their offspring go into cages with an amber label, and their offspring go into breeding cages with a red label. No offspring from red-label cages are used for breeding. Four generations of multiplication means that even a poorly breeding strain can produce large numbers of experimental animals.

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