Definition of an inbred strain. According to the official rules:
“A strain shall be regarded as inbred when it has been mated brother x sister (hereafter called b x s) for twenty or more consecutive generations (F20), and can be traced to a single ancestral breeding pair in the 20th. or a subsequent generation. Parent x offspring matings may be substituted for b x s matings provided that, in the case of consecutive parent x offspring matings, the mating in each case is to the younger of the two parents. Exceptionally, other breeding systems may be used, provided that the inbreeding coefficient achieved is at least equal to that at F20”.
Effects of inbreeding
Inbreeding leads to increased homozygosity, so that after 20 generations the probability of homozygosity at any gene locus is more than 98%. Ensuring that all animals can be traced to a single ancestral breeding pair in the 20th. or a subsequent generation means that all animals will also be genetically identical, with all parallel lines being eliminated. Note that the definition of an inbred strain is purely operational, it does not depend on any genetic test to ensure that the strain really is inbred.
Inbreeding alsoincreases the total genetic variance, but this is all expressed as differences between parallel lines. The within-line variation is reduced. The increased variation is due to the “uncovering” of recessive genes, leading to a much increased range of phenotypic characteristics between strains. Thus a collection of 2-3 inbred strains will usually vary much more than animals from a single outbred stock.
Isogenicity: all individuals are genetically identical
In an isogenic strain every individual is genetically identical. This means that a single individual can be genotyped at some locus, and this will genotype the whole strain. A profile of the genes present in a strain can be built up as a result of the work of scientists throughout the world.
New colonies of isogenic strains can be established from a single breeding pair, which will carry all the genes present in an existing colony. This means that investigators around the world can work on strains which are virtually genetically identical apart from some genetic drift due to new mutations if the colonies have been separated for several generations (see long-term stability). This is in strong contrast to outbred stocks in which, unless a new colony is established with at least 25 breeding pairs, there may be substantial genetic drift when setting up a new colony.
Individuals within a strain are histocompatible. Tissues, cells and organs can be transplanted from one individual to another without immunological rejection. F1 hybrids will accept grafts from both parental strains as well as other individuals of the same cross.
Inbred strains (but not F1 hybrids) are homozygous at virtually all loci (with a few exceptions, see below). As a result, they breed true. They do not carry “hidden” recessive genes which may cause confusion in any study involving breeding where there may be unpredictable production of homozygotes.
However, a strain is never completely homnozygous. Among the 30,000 or so loci there will always be a few (typically about 5-10 loci) which are segregating due to recent mutations, and after only 20 generations there will also be some loci which still segregate as a result of residual heterozygosity which has not yet been fixed by the inbreeding. Also, some non-coding mini-satellite loci are highly mutable, so may also be segregating within the strain. But for most practical purposes inbred strains can be regarded as being fully homozygous.
One result of homozygosity is that some deleterious recessive genes will become fixed within each strain, leading to inbreeding depression. Sometimes this is so severe that the strain dies out in the early generations of inbreeding. This inbreeding depression is seen most clearly in terms of poor breeding performance. Whereas outbred stocks may produce litters of 12 or more pups, inbred strains typically produce half this number. This inbreeding depression is a serious handicap in any study involving breeding. Thus investigators developing genetically modified animals are sometimes compelled to outcross the stock in order to obtain viable offspring. In such cases it is strongly recommended that the transgene is backcrossed to an inbred genetic background as soon as possible in order to fix its characteristics.
F1 hybrids, the first generation cross between two inbred strains, are isogenic but are also heterozygous at all loci at which the parental strains differ. This results in hybrid vigour. They are tend to be strong, vigorous, and breed well. However their offspring will be genetically segregating at many loci.
The total observed (phenotypic) variability of each character such as body weight, lifespan, enzyme activity etc. is composed of both genetic and non-genetic (“environmental”) components . However, within an isogenic strain the genetic varability is zero because all individuals are genetically identical, so the total variability is reduced. The extent of the reduction depends on the heritability of the character; the ratio of genetic variance to genetic plus environmental variance. Strongly inherited characters in which the environmental influences are relatively unimportant will be much more uniform in isogenic strains. The variation of weakly inherited characters with a strong environmental component will be reduced to a lesser degree.
It is usually important to be able to check whether the strain of mice or rats being used in a study is authentic and has not been genetically contaminated. This is relatively easy with isogenic strains as the genotype at a range of marker loci is known and published. The most widely used markers at present are microsatellites, but single nucleotide polymorphisms (SNPs) may become more widely used in the future.
Unfortunately, such information is not available for outbred stocks where any genetic quality control program would need to monitor both invariant loci in order to detect genetic contamination, and segregating loci in order to detect any changes in gene frequency which would be the result of genetic drift. At present it is not even possible to distinguishing between Wistar and Sprague-Dawley rats, the two most widely used stocks, although it is highly probable that stocks from different breeders are genetically different.
Inbred strains will not change as a result of selective breeding or further inbreeding, say as a result of restricted population size, so they stay genetically stable for long periods of time. Genetic contamination can be identified by routine genetic quality control using genetic markers.
The only way in which they can change is as a result of new mutations. Although these are relatively rare they do lead to substrain drift, as recognised in the genetic nomenclature. Many of the more common strains have been split into a number of branches, sometimes for many generations. Inevitable new mutations occur, and when they do so there is about a 1/4 chance that they will become fixed. Further substrain drift can be eliminated by maintaining banks of frozen embryos
Each isogenic strain is unique with its own specific set of characteristics. While this is also true of outbred stocks, the genetic stability and ability to identify each strain genetically means that it is possible to build up a profile of the phenotypic characteristics of each isogenic strain. This information can be used in choosing appropriate strains for a particular study, such as toxicity testing, and in interpreting the results of such studies.
Obviously care needs to be taken in choosing strains suited to a particular application. The above lists may be used to eliminate those strains which are clearly unsuitable. For example, few people except cancer research workers want to work with strains having an exceptionally high incidence of cancer. Where there is no information on which strain is likely to be most useful a small multi-strain study using small numbers of animals of 5-10 inbred or F1 hybrid strains might be appropriate (see multi-strain studies).
The use of a single strain is often appropriate in fundamental research, where the main aim might be to have a strain which is highly repeatable and shows a good response to the type of experimental treatment which is to be given. However, in screening experiments such as in toxicity testing a better strategy might be to use small numbers of several strains in a factorial experimental design as described under multi-strain studies.
Inbred strains tend to be sensitive to adverse environmental conditions so need to have good quality housing and they should be free of disease. They also tend to be more sensitive to experimental treatments than outbred stocks, which is a positive advantage. F1 hybrids tend to be even more uniform than inbred strains and are more “buffered” against adverse environments, hence in theory they may be less sensitive to an experimental treatment although there seems to be little objective evidence that this is the case.
In conclusion, isogenic strains can be regarded as the nearest approach to a “pure” reagent, somewhat analogous to pure analytical grade chemicals that are used in most research. For species such as the mouse and rat it makes no sense to use “impure” outbred animals in research when isogenic strains are readily available.