The use of inbred animals in experimental set ups in medical research

An introduction to animal models

The use of non-human animals in the study of human biology, health, disease and treatment has existed for millennia, with the first recorded experimentation through comparative science occurring in the 6th century BCE in Ancient Greece, where Alcmaeon of Croton discovered that the brain was the centre of intelligence and sensory integration through studies on dogs (Ericsson, Crim & Franklin, 2013). In the past century, the role of comparative biology has become vital to practically all fields of medical research: from immunology to neuroscience; behavioural studies to the research of infectious disease (Ericsson, Crim & Franklin, 2013). In the development of modern pharmaceutical drugs, animal models are utilised in the pre-clinical stage of trials to assess basic drug safety, efficacy, and pharmacokinetic mechanisms and parameters in vivo before progressing to human clinical trials (Belma et al., 2019).

A significant benefit of using laboratory populations of animal models in medical research is the level of control over an in vivo study this affords the researchers: in addition to facilitating consistent measurement and observation, environmental factors can be controlled and variables eliminated (or minimised) to a degree that simply would not be possible in a clinical or wild setting (Phillips & Roth, 2019), while causal factors of phenotypic differences among the subjects can be more confidently determined thanks to a largely genetically homogenous test population (Chebib et al., 2021), again eliminating variables in a way that would not be possible in a human clinical or wild animal setting. Being able to vary parameters of interest over a stable and consistent genetic base is key in ensuring experimental reproducibility (Zutphen, Baumans & Beynen, 2001), and allows researchers to measure and analyse difference while being able to rule out genetic variance (Chebib et al., 2021). These genetically stable and homogenous populations of animal models are achieved in the form of inbred strains.

History, definition and generation of inbred animals

The first scientific attempt to create a genetically uniform animal population involved an inbreeding experiment that took place in 1906 on guinea pigs (Cavia porcellus) (Wright, 1960) – fascinatingly, two of the Cavia porcellus strains from the original 1906 experiment are still in use in research today (Casellas, 2011)! Throughout the 20th century, work was done to generate inbred lines of a wide variety of species, from mammals (including the dog, pig, rat and Syrian hamster), birds (including the chicken, duck and quail) and fish (including the guppy, trout and zebra fish) to reptiles (the rattlesnake) and amphibians (the Xenopus genus of frog), in addition to invertebrates (such as C. elegans and Drosophila sp.) (Casellas, 2011). Today, the most widely used inbred strains are those of Mus musculus, the house mouse (Engber, 2011), with the first line (DBA – diluted, brown and non-agouti) created in 1909 (Holmes, 2003).

An inbred mouse strain is generally defined according to two core requirements: (i) at least 20 consecutive generations of (full) sibling mating have occurred; and (ii) all members are descended from a single pair of individuals (Carter et al., 1952). Theoretically, these specifications assure an inbreeding minimum level of 98.6%, or less than 2% of genetic variance from the base generation, ideally resulting in an almost completely isogenic, phenotypically uniform, stable population (Festing, 1979). In reality, 20 generations of full-sib inbreeding does not result in perfect isogenicity, with a low level of genetic variability persisting: in the 20th generation, over 200 polymorphic loci still exist (Bailey, 1982a). However, in theory, this is reduced asymptotically to zero through successive rounds of inbreeding (Wright, 1934) – and considering that many current commercially offered inbred mouse strains have significantly surpassed 20 generations (the massively popular C57BL/6J strain from The Jackson Laboratory had undergone 226 generations as of early 2010), it could be concluded that inbreeding is theoretically ‘complete’ in such commercial strains (Casellas, 2011).

The typical industry standard scheme used by commercial mouse breeders is of a pyramidal nature: each inbred strain is cryopreserved (pre-implantation embryos or sperm are frozen in liquid nitrogen (Northwestern University Center for Genetic Medicine, 2024)) forming what is known as the colony nucleus, with these embryos/sperm periodically used to generate an expansion colony from which, according to demand, mice can be transferred to production colonies. From these, they are ultimately delivered to research laboratories (Chebib et al., 2021) (see Figure 1).

Use of inbred mice in medical research

As mentioned, animal models play vital roles in the research of human health and disease, and the development of new pharmaceuticals. Due to the ability of inbred animals to provide researchers with a uniform genetic base from which to apply treatments and record observations, without the interference of genetic variability that would be found in a clinical setting or a wild population of animals, they play a key role in facilitating experimental reproducibility and enabling confident identification of causal factors (Zutphen, Baumans & Beynen, 2001). Thanks to these benefits, the use of isogenic inbred mice in modern medical research is prolific across a diverse range of fields, with specific commercial strains offered for different research focuses.

In the study of breast cancer, the second highest cause of cancer deaths globally (Brenner et al., 2020), mammary specific polyomavirus middle T antigen overexpression mouse model (MMTV-PyMT) strains are the most commonly used and have facilitated research into several aspects of the disease from initiation, histology and metastasis to, more recently, cancer immunotherapies (Attalla et al., 2021). Females of this transgenic strain develop malignant mammary tumours, with high penetrance and early onset (The Jackson Laboratory, 2024). Despite PyMT not being a human oncogene, it mimics receptor tyrosine kinase signalling pathways commonly dysregulated in human breast cancers, allowing researchers to study critical oncogenic mechanisms and potential therapeutic interventions in a clinically relevant model (Attalla et al., 2021).

Genetic stability and limitations of inbred mice

As discussed, the primary advantage of using inbred mouse strains is the isogenic nature of their populations, eliminating interference from genetic variation and providing a uniform genetic and phenotypic base for experimentation, which is key for ensuring reproducibility (Zutphen, Baumans & Beynen, 2001) – a key principle in the scientific method (Godfrey-Smith, 2003). Following the hundreds of generations of full-sib mating that many of the main commercial strains have undergone, individuals are expected to be homozygous across their entire genome (Silver, 1995). However, this neglects to consider the realities of spontaneous mutations (Chebib et al., 2021), heterozygote selection, and the possibility of contamination (Casellas, 2011). In fact, numerous studies have reported varying levels of genetic heterogeneity among inbred strains (Lynch, 1988), in various species from rats (Smits et al., 2004) to zebrafish (Guryev et al., 2006).

Mutation

The vulnerability of inbred mouse strains to mutation was first evidenced through a single mutation with significant phenotypic effects (Lord, 1929), and similar such mutations with obvious effects on phenotype, such as coat colour or dramatic behaviour change, continue to be observed in modern stocks to this day, even giving rise to new inbred strains (Casellas, 2011). However, more challenging are mutations with less obvious phenotypic consequences, which can easily go unnoticed without whole genome sequencing, specific research, or sometimes just extremely good fortune – an example of the latter is the discovery of the mini-muscle mutation, which causes a reduction in the muscle mass of mice’s hind limbs (Hartmann et al., 2008), and was coincidently observed during a detailed dissection of 14th generation individuals selected for voluntary wheel running activity (Houle-Leroy et al., 2000). It follows that such phenotypically harder-to-detect mutations are less likely to be identified and thus less likely to be removed from the breeding stock.

Heterozygote selection

The prediction of the aforementioned trend of genetic heterogeneity reducing asymptotically to zero through successive rounds of inbreeding  was formulated based upon the assumption of equal reproductive fitness among members of the same generation (Hartmann et al., 2008). However, if more than one breeding pair exists for a given generation, individuals that are more heterozygous may exhibit greater reproductive fitness, thus contributing more to the next generation than more homozygous individuals, in a phenomenon known as heterozygote selection: individuals with greater heterozygosis having a reproductive advantage over homozygotes, countering the desired homozygote fixation in an inbred strain (Bailey, 1982b). This phenomenon could also cause multi-generational persistence of the previously discussed spontaneous mutations.

While the stipulations for the definition of inbred mice dictate that members of a strain must be derived from a single pair in generation 20 or later (Carter et al., 1952), no restriction is placed on the number of breeding pairs in the generations that follow, meaning heterozygote selection is indeed possible. As such, it has been suggested that the trend of heterogeneity reduction asymptotically to zero (Wright, 1934) should instead be viewed as an upper bound for within-generation inbreeding (Casellas, 2011).

Contamination

While modern inbred mouse breeding populations are highly controlled, the possibility of unintended mating between mice from different strains has been documented (Nitzki et al., 2007), resulting in an introgression of heterogeneity. The heterozygosity caused by this can vary from a single nucleotide position to hundreds or even thousands of genes across all chromosomes, depending on how genetically distinct the two mating strains are.

This was evidenced in a 2021 study that sequenced the whole genomes of pairs of mice from four commonly-used inbred laboratory strains, that found that the strain obtained directly from the colony nucleus (C3H in this case) had a six-fold reduction in the number of segregating sites compared to the strains obtained from production lines (Chebib et al., 2021). Finally, while commercial breeders perform regular quality controls to ensure genetic homogeneity, this tends to use single nucleotide polymorphism (SNP) panels which only test a very small percentage of the overall genome (for example, just 27 SNPs tested at Jackson Labs, 48 at Envigo, and 96 at Taconic Biosciences (Chebib et al., 2021)). Ultimately, whole genome sequencing would be required to be absolutely confident in the complete isogenicity of individuals within a strain, which is (currently) not financially realistic from a commercial standpoint.