Getting Started with Diversity Outbred Mice

What are genetically diverse mouse populations and how are they used in research?

Inbred strains of laboratory mice have made significant contributions to biomedical research. However, for complex traits and diseases, deeper insight can be gained by working with genetically diverse mouse populations. The addition of genetic diversity to mouse-based studies improves the range and scope of their validity. Moreover, diverse mouse populations offer genetic and phenotypic variation that better recapitulates the human population, while providing added power for genetic mapping, as well as extensive tools and methodologies for experimental manipulation and validation.

Recombinant inbred (RI) and outbred populations of laboratory mice have been developed as research tools for complex trait and disease research. To create a panel of RI lines, two inbred strains are typically crossed to create F2’s, and F2’s are subsequently inbred through sibling intercrosses. The BXD recombinant inbred lines are a widely utilized panel that captures the allelic diversity resident in the two parental inbred strains, C57BL/6J and DBA/2J. To obtain greater allelic diversity, additional parental inbred strains can be added to these crosses. For example, the multi-parent Collaborative Cross (CC) RI lines were created through intercrossing and subsequent inbreeding of eight parental inbred strains. The CC lines increase mapping power, but like other RI panels retain the advantages of inbred mouse strains, including reproducible genomes. Derivatives of RI strains can be created through randomly intercrossing between RI lines. The Diversity Outbred (DO) mice were created from incipient CC RI lines. The DO population is genetically heterogeneous and therefore more closely matches the genetic structure of human populations. This diversity is particularly useful for measuring dose response across a population, high-resolution mapping of genes and quantitative trail loci (QTLs), and investigating genetic and environmental interactions that contribute to complex disease susceptibility, onset and progression, as well as behavioral phenotypes and drug response.

What are some of the genetically diverse mouse populations?

BXD

The BXD recombinant inbred (RI) lines were originally made for mapping highly penetrant Mendelian traits (Bailey et al., 1971, Taylor et al., 1973), but they were eventually adopted for the analysis of complex traits (Gora-Maslak et al. 1991). As a panel, these lines capture the allelic diversity resident in the two parental inbred strains, C57BL/6J and DBA/2J. Their main advantage relative to F2 crosses and heterogenous stocks is that each unique genotype (genetic individual) is represented by a stable inbred strain that can be replicated in large numbers — essentially a sexually reproducing clone. RIs are therefore an excellent resource for studies that benefit from replication across individuals (e.g. dosing and toxicity studies of genotypes) or across environments (i.e. studies on G×E), and for the gradual assembly of deep phenome data that can be used in G2P ((what is this?)) analysis. In mice, there are now sufficient numbers of RI strains to allow for comparatively precise and well-powered QTL mapping.

A number of RI panels are available from The Jackson Laboratory (https://www.jax.org/mouse-search/?stockType=Recombinant%20Inbred%20(RI)), including the BXD RI set (https://www.jax.org/mouse-search/?straingroup=BXD%20Strains)

Collaborative Cross

The Collaborative Cross (CC) is a large, multiparental, recombinant inbred (RI) strain panel that was created through rapid and random mixing of the genomes of eight founder strains to create independent breeding lines (Churchill et al. 2004). Five classical inbred strains (A/J, C57BL/6 J, 129S1/SvImJ, NOD/LtJ, NZO/H1LtJ) and three wild-derived strains (CAST/EiJ, PWK/PhJ, and WSB/EiJ) were selected as the eight parental (founder) strains of this cross. Analysis of the allelic variation in mouse inbred strains demonstrates that the eight CC founder strains capture on average 90% of the known allelic diversity across all 1-Mb intervals spanning the entire house mouse genome (Roberts et al. 2007), including the three major Mus subspecies, domesticus, musculus, and castaneous.

The founder inbred strains of the Collaborative Cross are available through The Jackson Laboratory, A/J (000646), C57BL/6J (000664), 129S1/SvImJ (002448) NOD/ShiLtJ (001976)  NZO/HiLtJ (002105), CAST/EiJ (000928), PWK/PhJ (003715), and WSB/EiJ (001145).

The Collaborative Cross lines are available from The University of North Carolina and The Jackson Laboratory (hyperlink: https://www.jax.org/mouse-search/?straingroup=Collaborative%20Cross)

Diversity Outbred

The Diversity Outbred (DO) (Svenson et al., 2012) population is a heterogeneous stock derived from 160 incipient Collaborative Cross lines (Collaborative Cross Consortium, 2012). Each DO mouse is a unique individual with a high level of allelic heterozygosity, and the DO population provides an effectively unlimited source of novel allelic combinations. The current generation, G32, harbors sufficient recombination events to provide sub-Mb mapping resolution across most regions of the genome. Mapping resolution will continue to improve with each successive generation. 

DO mice are available from The Jackson Laboratory (hyperlink: https://www.jax.org/strain/009376)

Related materials:

• Developing the Laboratory Mouse as a Tool for Systems Genetics, Gary Churchill, presentation to the New York Academy of Sciences, September 15, 2011
• Diversity Outbred Mice MiniCourse (online)

What should be taken into consideration when designing a study? 

• Cost and availability of resources (strains, hybrids, cases)
• Phenotype diversity, heritability, and genetic architecture
• Marker density, mapping precision, and power
• Availability of sequencing data, inaccessible regions of the genome
• Complexity of QTL intervals (if known)
• Population structure, admixture, and appropriate choice of analytic methods
• Depth of genetic, omics, and phenome data resources
• Robustness, replicability, extensibility (G×E), and translatability
• What mapping resolution will be sufficient to provide validation

How do I choose a genetic reference population for my research? 

BXD mice

Advantages

• Enable the simple aggregation of phenotypic data across time and treatment conditions.
• The lower genetic diversity of the BXD relative to multi-parent populations provides more power per allelic class in a similar sized mapping sample.
• The more recently created BXD strains provide unbiased correlation or independence of the strains relative to conventional inbred strains.
• Because the BXD strains are inbred, it is possible to characterize treatments and developmental endpoints in a genome-matched population.
• BXD strains can be intercrossed to create hybrid RIX lines to reveal effects attributable to heterozygosity.
• The replicability within strain enables high-precision estimation of phenotypic endpoints.

Considerations

• There are BXD populations harboring recombinant chromosomes that differ in their frequency and distribution, which can QTL intervals (see Peirce et al., 2004); known recombination cold spots can also limit QTL interval size.
• The load of polymorphisms within an interval may be an about 6-fold lower than that of the corresponding interval in the CC or DO stock, and thus the number of viable candidate genes may be reduced.
• Only a fraction of all known polymorphisms segregate in BXDs. For example, the BXD family segregates for a total of ~5.2 million sequence variants—about 44% of common variants among standard inbred strains (Roberts et al. 2007).
• Some strains possess fixed spontaneous mutations that have been documented.

Collaborative Cross mice

Advantages

• CC strains enable the simple aggregation of phenotypic data across time and treatment conditions.
• The recombination load (the crossover probability) of CC strains is 1.75 times higher than that of BXD.
• The high genetic diversity of the CC provide an expanded phenotypic range relative to the BXD population.
• The carefully structured breeding history of the CC provides unbiased correlation or independence of the strains relative to conventional inbred strains.
• Because the CC strains are inbred, it is possible to characterize treatments and developmental endpoints in a genome-matched population.
• The replicability within strain enables high-precision estimation of phenotypic endpoints.
• CC strains can be intercrossed to create hybrid RIX lines to reveal effects attributable to heterozygosity.
• Phenotypically extreme individual CC strains serve complex polygenic disease models.

Considerations

• The relatively small number of CC strains limits precision for genetic mapping except for traits with QTL that have large effect sizes.
• Small litter sizes make some lines difficult to maintain or obtain.
• Some strains possess fixed spontaneous mutations that have been documented.

Diversity Outbred mice

Advantages

• They reveal dominance and epistasis attributable to heterozygosity and randomized multi-locus combinations.
• More advanced generations capture more recombination events and therefore can resolve QTLs with high precision.
• The high genetic diversity among parental strains ensures that phenotypes will be highly variable and that most regions of the genome will be polymorphic.
• They have excellent breeding performance.

Considerations

• The allelic diversity will reduce statistical power to detect effects of rare variants and their epistatic actions; for such cases, consider using lower complexity populations such as the BXDs.
• DO animals are genetically unique. This means that it is more difficult to acquire phenomes for these types of resources or to use them as effectively in G×E studies.
• Some strains carry fixed spontaneous mutations that have been documented.
• The number of animals used will likely need to be greater than studies using inbred strains to achieve sufficient statistical power.

Use cases for Diversity Outbred mice

Diversity outbred mice identify population-based exposure thresholds and genetic factors that influence benzene-induced genotoxicity (toxicology, exposure threshold)

• Used DO mice to model genotoxic responses to environmental benzene
• Demonstrated reproducible responses using two independent cohorts of 300 DO animals each 
• Observed dose-dependent increase in benzene-induced chromosomal damage and determined a benchmark concentration limit an order of magnitude below the value estimated using a single inbred mouse strain
• Identified a locus containing two overexpressed sulfotransferases on chromosome 10 that inversely correlated with genotoxicity
• Identified the sulfotransferase genes to follow up for additional insight into benzene-induced genotoxicity
• DO mice displayed inter-individual variation in toxicity response that reflected human response ranges and provided a more sensitive threshold measurement than that obtained using an inbred strain

Genetic background influences susceptibility to chemotherapy-induced hematotoxicity (pharmacology, gene mapping)

• Used DO mice to identify genomic loci that influence chemotherapy-induced hematotoxicity, a life-threatening side-effect
• Dosed with one of three chemotherapy drugs with different modes of action–doxorubicin, cyclophosphamide, docetaxel–and observed a distinct effect on the underlying genetic architecture of hematotoxicity
• Generated data from 379 DO mice (191 females, 188 males) dosed with doxorubicin, 191 DO mice (97 females, 94 males) dosed with cyclophosphamide, and 154 DO mice dosed with docetaxel (85 females, 69 males) 
• Observed neutropenia levels and variation that recapitulated human patient responses
• The genetics underlying hematotoxicity differed between drugs, with a different locus identified for chemotherapy-induced neutropenia with each drug tested
• For doxorubicin, changes in cell counts was mapped to alleles of ATP-binding cassette B1 on chromosome 5, with presence of functional Abcb1b positively correlated with resistance to neutropenia.
• The doxorubicin finding was further investigated in knockout inbred mice
• The large genetic diversity offered by the DO mice allowed for sampling a larger number of genes and investigation of the toxicity of chemotherapy drugs in a whole-body system that include complex interactions between organs

High‐precision genetic mapping of behavioral traits in the diversity outbred mouse population (gene mapping)

• Behavioral testing of DO mice (N=283, male and female) used open-field, light–dark box, tail-suspension and visual-cliff avoidance tests to generate 38 behavioralmeasures
• Identified multiple quantitative trait loci (QTLs) for behavioral traits with support intervals ranging from 1 to 3 Mb in size
• QTLs identified including both previously published loci and novel loci for anxiety and activity related behaviors 
  
• Combining the founder allelic effects with whole genome sequence data further narrowed the positional candidates for a majority of QTLs
• Most QTL effects were explained by a single founder allele, but complex allelic patterns were also detectable
• The increased genetic diversity in the DO displayed a wide spectrum of behavior extending far beyond that of historical genetic mouse populations
• The results indicate the DO's value for comparatively fast, cost-effective, high precision QTL mapping of behavioral traits