Pathology of Genetically Engineered and Other Mutant Mice. Группа авторов

for identifying population outliers/rare susceptibility in toxicology and other types of studies. One big advantage of outbred stocks is that fecundity, reproductive efficiency, is usually quite high compared to inbred animals. This translates to lower costs to produce large numbers of animals in a short period of time. The major disadvantage is that because of the high phenotypic variability between individuals, large numbers need to be used to generate valid results, and the colony size and breeding schemes necessary to prevent inbreeding are large and more complex. This variability severely limits the ability to do highly refined research to ask very specific genetic and environmental questions about effects on disease or other phenotypes. But if the goal is to look for rare events, such as adverse drug reactions, these types of mice might better reflect the response in humans. This depends, of course, on breadth of alleles in the starting population and the rigor with which the outbred population is maintained. Defined outbred stocks are closed colonies, and inadvertent selection for fecundity, size, or health are selective pressures that reduce the genetic diversity of the population, as is any passage through a genetic bottleneck such as can occur during rederivation.

Table 3.10 Outbred mouse stocks.

AdvantagesHigh genetic and phenotypic variabilityCan be more reflective of the general outbred human populationRobust (high fecundity)Inexpensive

DisadvantagesLarge numbers needed for analysisHigh variability in results

UsesWidely used in all types of researchModels for human disease with variability between patients (population outliers)

One of the most commonly used outbred stocks is Crl:CD1 mice from Charles River Laboratories. This and other related “Swiss”‐derived stocks originated from seven females and two males were sent from a non‐inbred albino stock of Dr. Andre de Coulon at Centre Anticancereux Romand, Lausanne, Switzerland to Clara J. Lynch at Rockefeller University in the United States in 1926. These mice were further developed into the Hauschka Ha/ICR stock, which was initiated in 1948 at the Institute for Cancer Research (ICR) in Philadelphia. In 1959, a colony of mice, then maintained at Roswell Park Memorial Institute, were send to Charles River designated as HaM/ICR (https://www.criver.com/sites/default/files/resources/CD‐1IGSMouseModelInformationSheet.pdf). Today on the Charles River website you will find various trade names for different colonies of these mice including CD1‐Elite (SOPF) or CD1‐Elite Mouse. However, the correct formal nomenclature for the stock is Crl:CD1. While it is commonly used as an outbred stock, the genetic diversity from the small number of albino progenitors used and variability of sublines passed between institutions make this not the best example of an outbred stock.

      Fzt:DU provides a better example of an outbred stock [33]. This population was generated by crossing four other outbred stocks and four disparate inbred strains together to produce eight founder populations of 15 litters and then a careful breeding rotation was followed to maximize the allele frequencies in the population.

      Various breeding strategies have been reported in the literature for maintaining maximal genetic diversity in an outbred stock and all require a large population with a set rotation of breeding interactions. Random breeding places selective pressures on the population, most notably for a preference of increased fecundity, but has been used in some outbred stocks, and sibling breeding must be avoided. The nomenclature for outbred stocks begins with the laboratory code of the researcher who bred it followed by a colon followed by upper case stock name of two to four letters representing the population. Some outbred populations have been generated around a particular mutation in order to assess that phenotype in a diverse genetic setting. J:NU is an outbred stock bred around the nude (Foxn1^nu) mutation. Crl:SKH1‐Hr^hr is an outbred stock commonly used for UV light carcinogenesis studies because they have a high frequency for developing aggressive squamous cell carcinomas compared to other inbred strains carrying the hairless mutation [23]. The nomenclature (Crl:SKH1‐Hr^hr) again reflects the breeder (Crl), outbred strain (SKH1), and the specific allelic mutation Hr^hr.

Diversity Outbred (DO) Mice

      While there is genetic diversity in the outbred stocks described above, over time the genetic diversity will decrease without a very large colony maintained by a careful breeding scheme to avoid inbreeding. In addition, because each mouse is unique, there are no relevant control animals or sequenced reference genomes. These factors present significant difficulties in using outbred stocks. To address these issues and further expand genetic diversity, another approach was taken in recent years. As described above, the Collaborative Cross strains were developed using a mix of eight inbred strains that represented the most genetic diversity possible with existing inbred strains at the time. In the process of producing these mice, an alternative approach was developed in which 144 partially inbred Collaborative Cross strains, at generations ranging from F4 to F12, were maintained by randomized outcrossing, to create a novel population of mice, the Diversity Outbred stock, in which each individual mouse had an equal amount of DNA from all the progenitor strains, but all in a unique mix [34]. Each mouse is phenotypically and genetically different from the next and resulting in a huge variation in phenotypes. Large SNP genotyping arrays make it possible to phenotype large numbers of these mice that share the same phenotype and identify the candidate genes that might cause the problem [35–37].

      Nomenclature for the Diversity Outbred stocks follows that of outbred stocks, J:DO, where J is the breeder, The Jackson Laboratory in this example, and DO stands for Diversity Outbred.

Nomenclature for Normal and Mutated Genes

      Spontaneous mutations, including sequence variants, copy number variants, small indels, and multigenic chromosomal aberrations constantly arise in colonies but at relatively low rates so they are often missed unless a careful screening program is in place to identify them. This is called genetic drift and can be a major problem for large production colonies. There is also the real potential problem of genetic contamination, where strains are inadvertently mixed. To minimize and control these problems, two approaches are used in large production colonies: the Genetic Stability Program (GSP) (Methods for maintaining genetic stability of inbred animal strains (https://www.jax.org/jax‐mice‐and‐services/find‐and‐order‐jax‐mice/why‐jax‐mice/patented‐genetic‐stability‐program) US 7592501 [22 September 2009]; SG 119769; CN 200480023858.4 [23 June 2010]; AU [11 November 2010]; US 8110721 [7 February 2012]; JP 5072359 [31 August 2012]; USA 8552254 [8 September 2013]) and a genetic quality control program [37]. GSP essentially resets the genetic drift back to an arbitrary start point every ~5 generations by the use of a large pool of inbred embryos to replace the Founder stock. Genetic quality control programs regularly screen breeders at the top and sample the bottom of the production chain for a variety of molecular markers (SNPs) and phenotypes [38]. Newer methods, built around SNPs, utilize MiniMUGA Genotyping Arrays [38]. Prior to polymerase chain reaction (PCR), a variety of diagnostic screens (enzyme assays) and tail skin grafts were used (https://resources.jax.org/misc/jax‐handbook‐genetically‐standardized‐mice).

      Spontaneous mutations can be recessive, semi‐dominant, or dominant and can be true nulls, hypomorphic alleles, or gain‐of‐function mutations. Some result from retrovirus integration events, such as the hairless allele (Hr^hr) discussed above [39]. Radiation and chemical mutagenesis are also used to create mutations in mice without targeting specific genes, although specific phenotypes may be the focus. Genetically engineered mice can be created by transgenesis, recombineering, or nuclease mediated approaches. While many engineered mutations are called knockouts, not all are true nulls so this term has to be used carefully.

Spontaneous