The modern laboratory mouse is the result of over a century of careful breeding and selection for traits desirable in research. This effort has created hundreds of mouse strains with defined genetic backgrounds, each with unique characteristics such as coat color, behavior, metabolism, fecundity, immune function, and other physiological traits. As a result, certain strains may be suited for particular applications, while others may be inappropriate. This fact is well known to mouse geneticists, but is often underappreciated in the wider research community. Understanding how to select an appropriate strain and preventing genetic sources of variability are critical aspects to any in vivo research project.
Genetically, there are two major classes of laboratory mice: inbred and outbred. Inbred mice are genetically homogeneous and there is very little variation or heterogeneity within a pure inbred strain. Inbred strains are created by inbreeding or mating brother/sister pairs over at least 20 generations, leading to mice that are ~99% homozygous at all loci. During this inbreeding, many genetic variants become fixed and homozygous within the population, creating a specific and reliable genetic background on which to perform studies. The fact that all mice within an inbred strain are genetically similar may reduce experimental variability and increase study reproducibility, while enabling transplants between individuals without risk of rejection.
Outbred mice are bred specifically to maximize genetic diversity and heterozygosity within a population. In theory, no two individuals are genetically identical within an outbred stock, owing to rotational breeding schemes that intentionally prevent inbreeding. Outbred mice tend to be heartier, larger, and better breeders than inbred mice. They are often favored in toxicology studies, where the aim is to assess responses throughout a population of heterogenous individuals.
Although inbred mice are treated as genetically identical, errors in DNA replication and germline transmission invariably create genetic variation within an inbred strain. As a result, each mouse harbors around 60 new genetic variants. While this may seem insignificant compared to the size of the genome, these mutations will accumulate and may become fixed in the population, causing an inbred strain to drift genetically over time.
The result is that any inbred strain maintained for >20 generations will become genetically distinct from its parental strain. In the last 30 years, many animal vendors began to prevent this drift by “refreshing” breeding stock of inbred strains from archived cryopreserved embryos at defined intervals to reset this genetic clock and provide animals with a consistent genetic background over time. This has made genetic drift no longer an issue for most commercial mice, but the reality of drift still affects mouse breeding and its legacy is apparent in the numerous sub-strains available from different vendors.
There is no standard mouse strain; the selection of a particular inbred background should be based on research goals and the suitability of strain characteristics for experimental models. It is important to note that each inbred strain is represented by multiple different substrains, which may differ in genetic background and characteristics. Substrains are generally vendor-specific and specified in the complete strain nomenclature (e.g. C57BL/6NTac from Taconic Biosciences vs. C57BL/6J from the Jackson Laboratory). Experimental models should be validated on genetically defined inbred substrains, and care should be taken that genetic variability or contamination are not introduced through genetic drift or cross-contamination with other substrains.
One of the most common inbred strains in modern research is the C57BL/6 (“C57-black-6” or B6), preferred for many metabolism, obesity, diabetes, immunology, behavior, and oncology studies. It is a relatively good breeder and its genome has been sequenced.
Since the origin of this strain in the 1920s, at least 16 different C57BL/6 substrains have been developed, descended from colonies originating at the Jackson Laboratory (C57BL/6J substrains) or the National Institutes of Health (C57BL/6N substrains). All C57BL/6 substrains are immunologically similar, with the same cell surface markers and MHC haplotypes. Tissues, cells, and tumors of C57BL/6 origin can be engrafted into all C57BL/6 substrains successfully.
Figure 1
Although similar, there are differences between C57BL/6 substrains that should be considered. Notably, C57BL/6N substrains are more prone to weight gain and insulin resistance when maintained on high-fat diets. There are also genetic variants between C57BL/6 substrains that affect immune function, metabolism and behavior that should be considered in C57BL/6 substrain selection, as noted in Figure 1.
The strain background of genetically engineered models (GEMs) is a major issue. Genetic modifiers in different mouse strains can affect the penetrance and severity of phenotypes caused by knockouts and transgenes. In some cases, GEMs may be lethal on certain backgrounds, necessitating study in alternative strains.
When generating GEMs, the choice of strain is often limited by technical considerations. Homologous recombination requires efficient embryonic stem (ES) cells, only available from several inbred backgrounds. Many traditional knockouts were created on a 129/Sv background, from which the first efficient ES cells were created. Extensive backcrossing is required to generate congenic knockouts on a more suitable background for study, such as C57BL/6 or BALB/c. Insufficient backcrossing has caused many problems, as contaminating genes from other strains may significantly impair study interpretation.
To overcome background issues, many centers create GEMs using C57BL/6-derived ES cells, the vast majority of which are derived from the C57BL/6NTac substrain and used in major projects such as the Knockout Mouse Project (KOMP), the International Mouse Phenotyping Consortium (IMPC), and the European Conditional Mouse Mutagenesis Program (EUCOMM). Founders from these projects will possess the genetic profile of C57BL/6NTac mice, and care should be taken to not introduce genetic contamination through breeding with other C57BL/6 substrains.
Close attention to the genetic background of mouse models is critical to the success of your research project. Selecting an appropriate inbred strain and controlling for this while sourcing and breeding animals will increase model performance and experimental reproducibility.
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Images: Taconic