The diversity outbred mouse population

Mapping the Effects of Genetic Variation on Chromatin State and Gene Expression Reveals Loci That Control Ground State Pluripotency.

Multiparental populations (MPPs) encompass greater genetic diversity than traditional experimental crosses of two inbred strains, enabling broader surveys of genetic variation underlying complex traits. Two such mouse MPPs are the Collaborative Cross (CC) inbred panel and the Diversity Outbred (DO) population, which are descended from the same eight inbred strains. Additionally, the F1 intercrosses of CC strains (CC-RIX) have been used and enable study designs with replicate outbred mice. Genetic analyses commonly used by researchers to investigate complex traits in these populations include characterizing how heritable a trait is, i.e. its heritability, and mapping its underlying genetic loci, i.e. its quantitative trait loci (QTLs). Here we evaluate the relative merits of these populations for these tasks through simulation, as well as provide recommendations for performing the quantitative genetic analyses. We find that sample populations that include replicate animals, as possible with the CC and CC-RIX, provide more efficient and precise estimates of heritability. We report QTL mapping power curves for the CC, CC-RIX, and DO across a range of QTL effect sizes and polygenic backgrounds for samples of 174 and 500 mice. The utility of replicate animals in the CC and CC-RIX for mapping QTLs rapidly decreased as traits became more polygenic. Only large sample populations of 500 DO mice were well-powered to detect smaller effect loci (7.5-10%) for highly complex traits (80% polygenic background). All results were generated with our R package musppr, which we developed to simulate data from these MPPs and evaluate genetic analyses from user-provided genotypes.

Many studies of Mycobacterium tuberculosis infection and immunity have used mouse models. However, outcomes of vaccination and challenge with M. tuberculosis in inbred mouse strains do not reflect the full range of outcomes seen in people. Previous studies indicated that the novel Diversity Outbred (DO) mouse population exhibited a spectrum of outcomes after primary aerosol infection with M. tuberculosis Here, we demonstrate the value of this novel mouse population for studies of vaccination against M. tuberculosis aerosol challenge. Using the only currently licensed tuberculosis vaccine, we found that the DO population readily controlled systemic Mycobacterium bovis BCG bacterial burdens and that BCG vaccination significantly improved survival across the DO population upon challenge with M. tuberculosis Many individual DO mice that were vaccinated with BCG and then challenged with M. tuberculosis exhibited low bacterial burdens, low or even no systemic dissemination, little weight loss, and only minor lung pathology. In contrast, some BCG-vaccinated DO mice progressed quickly to fulminant disease upon M. tuberculosis challenge. Across the population, most of these disease parameters were at most modestly correlated with each other and were often discordant. This result suggests the need for a multiparameter metric to better characterize "disease" and "protection," with closer similarity to the complex case definitions used in people. Taken together, these results demonstrate that DO mice provide a novel small-animal model of vaccination against tuberculosis that better reflects the wide spectrum of outcomes seen in people.IMPORTANCE We vaccinated the Diversity Outbred (DO) population of mice with BCG, the only vaccine currently used to protect against tuberculosis, and then challenged them with M. tuberculosis by aerosol. We found that the BCG-vaccinated DO mouse population exhibited a wide range of outcomes, in which outcomes in individual mice ranged from minimal respiratory or systemic disease to fulminant disease and death. The breadth of these outcomes appears similar to the range seen in people, indicating that DO mice may serve as an improved small-animal model to study tuberculosis infection and immunity. Moreover, sophisticated tools are available for the use of these mice to map genes contributing to control of vaccination. Thus, the present studies provided an important new tool in the fight against tuberculosis.

Development of a new vaccine for tuberculosis (TB) to replace the BCG vaccine has been hampered by the lack of a preclinical vaccine model that accurately reflects the genetic heterogeneity of the human population. We previously tested both Collaborative Cross (CC) and Diversity Outbred (DO) mice, which collectively reflect the genetic diversity of an outbred population, as models for preclinical vaccine testing. Using both CC and DO mice, we compared the ability of two different strategies to enhance BCG-mediated protection against Mycobacterium tuberculosis (Mtb). BCG::ESX-1Mmar is genetically modified to express the esx-1 region of Mycobacterium marinum. In contrast, the BCG/ChAd:TB strategy relies on boosting BCG-induced immunity using a chimp adenoviral vectored vaccine expressing the mycobacterial antigens Ag85A, TB10.4, and RpfB administered by the intranasal route. Both BCG::ESX-1Mmar and BCG/ChAd:TB vaccination strategies significantly protect mouse strains that were not well protected by BCG. Although both vaccination strategies were associated with reduced bacterial growth in CC and DO mice, bacterial control did not correlate with survival. Both BCG::ESX-1Mmar and BCG/ChAd:TB induced immune signatures after Mtb challenge in the tested CC strains that were previously associated with protection in traditional inbred mouse strains. Our findings established the use of both CC and DO mice as robust platforms that incorporate host genetic diversity for the preclinical evaluation of TB vaccines.

Inbred mice exhibit strain-specific variation in susceptibility to atherosclerosis and dyslipidemia that renders them useful in dissecting the genetic architecture of these complex diseases. Traditional quantitative trait locus (QTL) mapping studies using inbred strains often identify large genomic regions, containing many genes, due to limited recombination and/or sample size. This hampers candidate gene identification and translation of these results into possible risk factors and therapeutic targets. An alternative approach is the use of multiparental outbred lines for genetic mapping, such as the Diversity Outbred (DO) mouse panel, which can be more informative than traditional two-parent crosses and can aid in the identification of causal genes and variants associated with QTL. We fed 292 female DO mice either a high-fat, cholesterol-containing (HFCA) diet, to induce atherosclerosis, or a low-fat, high-protein diet for 18 wk and measured plasma lipid levels before and after diet treatment. We measured markers of atherosclerosis in the mice fed the HFCA diet. The mice were genotyped on a medium-density single-nucleotide polymorphism array and founder haplotypes were reconstructed using a hidden Markov model. The reconstructed haplotypes were then used to perform linkage mapping of atherosclerotic lesion size as well as plasma total cholesterol, triglycerides, insulin, and glucose. Among our highly significant QTL we detected a ~100 kb QTL interval for atherosclerosis on Chromosome 6, as well as a 1.4 Mb QTL interval on Chromosome 9 for triglyceride levels at baseline and a coincident 22.2 Mb QTL interval on Chromosome 9 for total cholesterol after dietary treatment. One candidate gene within the Chromosome 6 peak region associated with atherosclerosis is Apobec1, the apolipoprotein B (ApoB) mRNA-editing enzyme, which plays a role in the regulation of ApoB, a critical component of low-density lipoprotein, by editing ApoB mRNA. This study demonstrates the value of the DO population to improve mapping resolution and to aid in the identification of potential therapeutic targets for cardiovascular disease. Using a DO mouse population fed an HFCA diet, we were able to identify an A/J-specific isoform of Apobec1 that contributes to atherosclerosis.

Hyperlipidemia, a risk factor for cardiovascular disease, is a complex trait regulated by a variety of dietary and genetic risk factors. Inbred mice, which exhibit strain‐specific variation in circulating lipoprotein levels, have effectively been used to model the genetic complexity of hyperlipidemia. Traditional quantitative trait locus (QTL) mapping studies using inbred strains often identify large genomic regions, containing many genes, due to limited recombination. This hampers candidate gene identification and translation of results into possible therapeutic targets. An alternative approach is the use of outbred strains for genetic mapping, such as the Diversity Outbred (DO) mouse panel recently developed by the Jackson Laboratory.Our current studies use the DO mice to map QTL for plasma lipid levels. DO mice were genotyped for 77,800 SNPs using the MegaMUGA array and founder haplotype states were predicted using a hidden Markov model. QTL mapping was carried out in R using a genetic model that incorporates reconstructed haplotypes and accounts for population structure. Our initial studies used 316 male and female DO mice fed a synthetic diet to map total cholesterol (TC) and triglyceride (TG) levels and identified a significant peak on Chromosome 1 (max LOD 8.02) and a suggestive peak on Chromosome 6 (max LOD 6.43) associated with TC. The Chromosome 1 QTL maps 1 Mb away from a well characterized gene regulating cholesterol (ApoA2). We identified two suggestive peaks on Chromosome 17 associated with TG. We have also measured plasma lipid levels and traits modeling atherosclerosis in an additional cohort of 300 female DO mice fed either a hyperlipidemia‐inducing diet or a control diet; mapping results are pending. Our studies are intended to demonstrate the value of the DO population to improve mapping resolution and aid in the identification of potential therapeutic targets for metabolic diseases.Grant Funding Source: Pilot funds from the Nutrition Obesity Research Center (P30DK056350) and the NHLBI (4R00HL102223)

Although humans are heterogeneous, evaluation of tumorigenesis and immunotherapy has relied on the use of inbred mouse strains, which may lead to biased results. In order to investigate the effect(s) of genetic background on tumor incidence and immune response, we have taken a unique new approach to identify genes that are associated with tumor onset time & tumor growth rate using Diversity Outbred (DO) mice. Specifically, we crossed DO female mice (J:DO, Jackson Labs) with BALB/NeuT (NeuT) mice to introduce a heterogeneous background in (BALBxDO) F1 NeuT mice. NeuT express transforming rat neu, the homologue of HER2, and develop spontaneous tumors in all 10 mammary glands between 14 and 19 weeks of age (woa). DO mice are generated by non-sibling crosses of 8 founder strains: A/J, C57BL/6J, 129S1/SvlmJ, NOD/ShiLtJ, NZO/HILtJ, CAST/EiJ, PWK/PhJ, WSB/EiJ to encompass greater than 90% of murine polymorphic alleles. The genetic makeup of each F1 mouse is defined using 143,600 chromosomal markers (mostly SNP) in the Giga Mouse Universal Genotyping Array. SNP analysis identifies the origin of quantitative trait loci (QTL), using the R/QTL package. The program reads the GigaMUGA genotyping results to perform haplotype reconstruction and determine the founder strain haplotype of each marker locus. DO F1 mice have been successfully used to identify the critical role of MHC-IB, and NK cells, in generation of HER2 Ab response following vaccination of HER2 tolerant mice (Wei et al., 2020). In (BALBxDO) F1 NeuT female mice most spontaneous mammary tumors develop over a wider range of time from 9 to 23 woa. QTL analysis of tumor onset time revealed QTLs in chromosomes (Chr) 1 and X with peak LOD scores of 7.7 (p = 0.01) and 8.6 (p < 0.05), respectively. Tumor growth rate was associated with a QTL in Chr 10. More specifically, PWK and CAST haplotypes in these QTLs were associated with early and faster growing tumors. About 10-15 genes in the 3 QTLs contain missense SNP alterations unique to PWK and CAST, and the genes have been associated with human breast cancer prognosis. As an example, Mid2 identified in ChrX QTL interacts with BRCA1, and is negatively associated with patient survival using the TCGA BRCA database. Single cell RNA sequencing revealed that Mid2 is expressed in NeuT mammary tumor tissue. Studies are ongoing to determine the functional significance of candidate genes in HER2/neu mammary tumorigenesis. DO F1 QTL combined with scRNA transcriptomic analysis opens important new opportunities to define critical genes in cancer biology and immunotherapy, with the potential for novel intervention and disease prevention. Citation Format: Jennifer Jacob, Kuang Wei, Joyce D. Reyes, Cong-Cong Yin, Elizabeth Rondini, Qing-Sheng Mi, James Granneman, Prahlad Parajuli, K Wagner, Wei-Zen Wei. A Diversity outbred (DO) mouse model reveals regulators of HER2-driven tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2920.

A strong predictor for the development of alcohol use disorder (AUD) is altered sensitivity to the intoxicating effects of alcohol. Individual differences in the initial sensitivity to alcohol are controlled in part by genetic factors. Mice offer a powerful tool to elucidate the genetic basis of behavioral and physiological traits relevant to AUD, but conventional experimental crosses have only been able to identify large chromosomal regions rather than specific genes. Genetically diverse, highly recombinant mouse populations make it possible to observe a wider range of phenotypic variation, offer greater mapping precision, and thus increase the potential for efficient gene identification. We have taken advantage of the Diversity Outbred (DO) mouse population to identify and precisely map quantitative trait loci (QTL) associated with ethanol sensitivity. We phenotyped 798male J:DO mice for three measures of ethanol sensitivity: ataxia, hypothermia, and loss of the righting response. We used high-density MegaMUGA and GigaMUGA to obtain genotypes ranging from 77,808 to 143,259 SNPs. We also performed RNA sequencing in striatum to map expression QTLs and identify gene expression-trait correlations. We then applied a systems genetic strategy to identify narrow QTLs and construct the network of correlations that exists between DNA sequence, gene expression values, and ethanol-related phenotypes to prioritize our list of positional candidate genes. We observed large amounts of phenotypic variation with the DO population and identified suggestive and significant QTLs associated with ethanol sensitivity on chromosomes 1, 2, and 16. The implicated regions were narrow (4.5-6.9 Mb in size) and each QTL explained ~4-5% of the variance. Our results can be used to identify alleles that contribute to AUD in humans, elucidate causative biological mechanisms, or assist in the development of novel therapeutic interventions.

Understanding how genetic variability shapes responses to environmental and developmental factors is critical for advancing translational neuroscience. However, most preclinical studies rely on inbred mouse strains that do not capture the genetic complexity of human populations. One key area of translational research focuses on identifying the neural and behavioral consequences of early life trauma. Rodent models of childhood neglect, such as maternal separation with early weaning (MSEW), have been used in isogenic mouse strains like C57BL/6J (B6) to identify behavioral domains and neural loci of deficits stemming from exposure to MSEW. To understand how genetic diversity may contribute to the outcomes produced by MSEW, and thus inform future studies on the topic, we utilized the Jackson Laboratory Diversity Outbred (DO) line, a population derived from eight founder strains that exhibit broad genetic and phenotypic heterogeneity. We first compared MSEW effects on social behavior in DO mice versus B6 mice, because we have previously found social behavior deficits in B6 mice with a history of MSEW. Indeed, we established that MSEW incited social motivation deficits in DO mice, in a sex-specific manner. We then expanded our investigation of DO mice to test MSEW-related changes in anxiety-like behavior, fear learning and expression, and reward-seeking. Results revealed that MSEW produces distinct, sex-specific phenotypes: female DO mice displayed reduced social motivation and elevated anxiety-like behavior, while male DO mice showed attenuated CS-evoked fear expression and diminished reward-seeking behavior. Additionally, immunohistochemical analysis revealed increased Fos expression in the paraventricular nucleus of the hypothalamus (PVN) in MSEW-exposed DO mice, both at baseline and following acute stress. These findings highlight the importance of incorporating genetically diverse models to better capture the nuances of early life adversity-related outcomes relevant to human populations.

Understanding how genetic variability shapes responses to environmental and developmental factors is critical for advancing translational neuroscience. However, most preclinical studies rely on inbred mouse strains that do not capture the genetic complexity of human populations. One key area of translational research focuses on identifying the neural and behavioral consequences of early life trauma. Rodent models of childhood neglect, such as maternal separation with early weaning (MSEW), have been used in isogenic strains like C57BL/6J (B6) to identify behavioral domains and neural loci of deficits stemming from exposure to MSEW. To understand how genetic diversity may contribute to the outcomes produced by MSEW, and thus inform future studies on the topic, we utilized the Jackson Laboratory Diversity Outbred (DO) line, a population derived from eight founder strains that exhibit broad genetic and phenotypic heterogeneity. We first compared MSEW effects on social behavior in DO mice versus B6 mice, because we have previously found social behavior deficits in B6 mice with a history of MSEW. Indeed, we established that MSEW incited social motivation deficits in DO mice, in a sex-specific manner. We then expanded our investigation of DO mice to test MSEW-related changes in anxiety-like behavior, fear learning and expression, and reward-seeking. Results revealed that MSEW produces distinct, sex-specific phenotypes: female DO mice displayed reduced social motivation and elevated anxiety-like behavior, while male DO mice showed attenuated CS-evoked fear expression and diminished reward-seeking behavior. Additionally, immunohistochemical analysis revealed increased Fos expression in the paraventricular nucleus of the hypothalamus (PVN) in MSEW-exposed DO mice, both at baseline and following acute stress. These findings highlight the importance of considering genetically diverse models to better capture the nuances of early life adversity-related outcomes relevant to human populations.

Understanding how genetic variability shapes responses to environmental and developmental factors is critical for advancing translational neuroscience. However, most preclinical studies rely on inbred mouse strains that do not capture the genetic complexity of human populations. One key area of translational research focuses on identifying the neural and behavioral consequences of early life trauma. Rodent models of childhood neglect, such as maternal separation with early weaning (MSEW), have been used in isogenic strains like C57BL/6J (B6) to identify behavioral domains and neural loci of deficits stemming from exposure to MSEW. To understand how genetic diversity may contribute to the outcomes produced by MSEW, and thus inform future studies on the topic, we utilized the Jackson Laboratory Diversity Outbred (DO) line, a population derived from eight founder strains that exhibit broad genetic and phenotypic heterogeneity. We first compared MSEW effects on social behavior in DO mice versus B6 mice, because we have previously found social behavior deficits in B6 mice with a history of MSEW. Indeed, we established that MSEW incited social motivation deficits in DO mice, in a sex-specific manner. We then expanded our investigation of DO mice to test MSEW-related changes in anxiety-like behavior, fear learning and expression, and reward-seeking. Results revealed that MSEW produces distinct, sex-specific phenotypes: female DO mice displayed reduced social motivation and elevated anxiety-like behavior, while male DO mice showed attenuated CS-evoked fear expression and diminished reward-seeking behavior. Additionally, immunohistochemical analysis revealed increased Fos expression in the paraventricular nucleus of the hypothalamus (PVN) in MSEW-exposed DO mice, both at baseline and following acute stress. These findings highlight the importance of considering genetically diverse models to better capture the nuances of early life adversity-related outcomes relevant to human populations.

Adult hippocampal neurogenesis-the generation of new neurons in the adult brain-declines with age, contributing to cognitive deficits in aging. While the majority of mammalian studies on neurogenesis have utilized inbred mouse strains, these models do not fully capture the genetic diversity of humans, limiting the translational relevance of their findings. The Diversity Outbred (DO) mouse model, a genetically heterogeneous population, provides a promising alternative to traditional inbred strains. In this study, we investigated how genetic diversity influences hippocampal neurogenesis by comparing neurogenesis in adult and aged Diversity Outbred (DO) mice with the commonly used C57BL/6J inbred strain. While both strains exhibited a decline in neurogenesis with age, DO mice showed significantly lower levels of neurogenesis compared to C57BL/6J mice, even in young adults. Additionally, we observed that the wild-derived CAST/EiJ strain, one of the eight founder strains in the DO model, contributed to this reduction in neurogenesis. Our findings highlight the importance of genetic diversity in neurogenesis research and suggest that the DO model may better represent human genetic diversity associated with age-related decline in neurogenesis.

Behavioral phenotypes revealed during reversal learning are linked with novel genetic loci in diversity outbred mice

Interindividual variation in voluntary ethanol consumption and ethanol response is partially influenced by genetic variation. Discovery of the genes and allelic variants that affect these phenotypes may clarify the etiology and pathophysiology of problematic alcohol use, including alcohol use disorder. Genetically diverse mouse populations, which demonstrate heritable variation in ethanol consumption, can be utilized to discover the genes and gene networks that influence this trait. The Collaborative Cross (CC) recombinant inbred strains, Diversity Outbred (DO) population and their 8 founder strains are complementary mouse resources that capture substantial genetic diversity and can demonstrate expansive phenotypic variation in heritable traits. These populations may be utilized to discover candidate genes and gene networks that moderate ethanol consumption and other ethanol-related traits. We characterized ethanol consumption, preference, and pharmacokinetics in the 8 founder strains and 10 CC strains in 12-hour drinking sessions during the dark phase of the circadian cycle. Ethanol consumption was substantially heritable, both early in ethanol access and over a chronic intermittent access schedule. Ethanol pharmacokinetics were also heritable; however, no association between strain-level ethanol consumption and pharmacokinetics was detected. The PWK/PhJ strain was the highest drinking strain, with consumption substantially exceeding that of the C57BL/6J strain, which is commonly used as a model of "high" or "binge" drinking. Notably, we found strong evidence that sex moderated genetic effects on voluntary ethanol drinking. Collectively, this research serves as a foundation for expanded genetic study of ethanol consumption in the CC/DO and related populations. Moreover, we identified reference strains with extreme consumption phenotypes that effectively represent polygenic models of excessive ethanol use.

Immune checkpoint inhibitors (ICI) have improved outcomes for a variety of malignancies; however, many patients fail to benefit. While tumor-intrinsic mechanisms are likely involved in therapy resistance, it is unclear to what extent host genetic background influences response. To investigate this, we utilized the Diversity Outbred (DO) and Collaborative Cross (CC) mouse models. DO mice are an outbred stock generated by crossbreeding eight inbred founder strains, and CC mice are recombinant inbred mice generated from the same eight founders. We generated 207 DOB6F1 mice representing 48 DO dams and demonstrated that these mice reliably accept the C57BL/6-syngeneic B16F0 tumor and that host genetic background influences response to ICI. Genetic linkage analysis from 142 mice identified multiple regions including one within chromosome 13 that associated with therapeutic response. We utilized 6 CC strains bearing the positive (NZO) or negative (C57BL/6) driver genotype in this locus. We found that 2/3 of predicted responder CCB6F1 crosses show reproducible ICI response. The chromosome 13 locus contains the murine prolactin family, which is a known immunomodulating cytokine associated with various autoimmune disorders. To directly test whether prolactin influences ICI response rates, we implanted inbred C57BL/6 mice with subcutaneous slow-release prolactin pellets to induce mild hyperprolactinemia. Prolactin augmented ICI response against B16F0, with increased CD8 infiltration and 5/8 mice exhibiting slowed tumor growth relative to controls. This study highlights the role of host genetics in ICI response and supports the use of F1 crosses in the DO and CC mouse populations as powerful cancer immunotherapy models.