Recombinant Inbred Panel Research Articles

Genome-wide screen identifies host loci that modulate Mycobacterium tuberculosis fitness in immunodivergent mice.

Genetic differences among mammalian hosts and among strains of Mycobacterium tuberculosis (Mtb) are well-established determinants of tuberculosis (TB) patient outcomes. The advent of recombinant inbred mouse panels and next-generation transposon mutagenesis and sequencing approaches has enabled dissection of complex host-pathogen interactions. To identify host and pathogen genetic determinants of Mtb pathogenesis, we infected members of the highly diverse BXD family of strains with a comprehensive library of Mtb transposon mutants (TnSeq). Members of the BXD family segregate for Mtb-resistant C57BL/6J (B6 or B) and Mtb-susceptible DBA/2J (D2 or D) haplotypes. The survival of each bacterial mutant was quantified within each BXD host, and we identified those bacterial genes that were differentially required for Mtb fitness across BXD genotypes. Mutants that varied in survival among the host family of strains were leveraged as reporters of "endophenotypes," each bacterial fitness profile directly probing specific components of the infection microenvironment. We conducted quantitative trait loci (QTL) mapping of these bacterial fitness endophenotypes and identified 140 host-pathogen QTL (hpQTL). We located a QTL hotspot on chromosome 6 (75.97-88.58 Mb) associated with the genetic requirement of multiple Mtb genes: Rv0127 (mak), Rv0359 (rip2), Rv0955 (perM), and Rv3849 (espR). Together, this screen reinforces the utility of bacterial mutant libraries as precise reporters of the host immunological microenvironment during infection and highlights specific host-pathogen genetic interactions for further investigation. To enable downstream follow-up for both bacterial and mammalian genetic research communities, all bacterial fitness profiles have been deposited into GeneNetwork.org and added into the comprehensive collection of TnSeq libraries in MtbTnDB.

Effects of isolation housing stress and mouse strain on intravenous cocaine self-administration, sensory stimulus self-administration, and reward preference

Sensory stimuli are natural rewards in mice and humans. Consequently, preference for a drug reward relative to a sensory reward may be an endophenotype of addiction vulnerability. In this study, we developed a novel behavioral assay to quantify the preference for intravenous drug self-administration relative to sensory stimulus self-administration. We used founder strains of the BXD recombinant inbred mouse panel (C57BL/6J, DBA/2J) and a model of stress (isolation vs enriched housing) to assess genetic and epigenetic effects. Following 10 weeks of differential housing, all mice were tested under three reward conditions: sensory rewards available, cocaine rewards available, both rewards available. When a single reward was available (sensory stimuli or cocaine; delivered using distinct levers), DBA/2J mice self-administered significantly more rewards than C57BL/6J mice. When both rewards were available, DBA/2J mice exhibited a significant preference for cocaine relative to sensory stimuli; in contrast, C57BL/6J mice exhibited no preference. Housing condition influenced sensory stimulus self-administration and strain-dependently influenced inactive lever pressing when both rewards were available. Collectively, these data reveal strain effects, housing effects, or both on reward self-administration and preference. Most importantly, this study reveals that genetic mechanisms underlying preference for a drug reward relative to a nondrug reward can be dissected using the full BXD panel.

Intravenous fentanyl self-administration in male and female C57BL/6J and DBA/2J mice

The genetic mechanisms underlying fentanyl addiction, a highly heritable disease, are unknown. Identifying these mechanisms will lead to better risk assessment, early diagnosis, and improved intervention. To this end, we used intravenous fentanyl self-administration to quantify classical self-administration phenotypes and addiction-like fentanyl seeking in male and female mice from the two founder strains of the BXD recombinant inbred mouse panel (C57BL/6J and DBA/2J). We reached three primary conclusions from these experiments. First, mice from all groups rapidly acquired intravenous fentanyl self-administration and exhibited a dose–response curve, extinction burst, and extinction of the learned self-administration response. Second, fentanyl intake (during acquisition and dose response) and fentanyl seeking (during extinction) were equivalent among groups. Third, strain effects, sex effects, or both were identified for several addiction-like behaviors (cue-induced reinstatement, stress-induced reinstatement, escalation of intravenous fentanyl self-administration). Collectively, these data indicate that C57BL/6J and DBA/2J mice of both sexes were able to acquire, regulate, and extinguish intravenous fentanyl self-administration. Moreover, these data reveal novel strain and sex effects on addiction-like behaviors in the context of intravenous fentanyl self-administration in mice and indicate that the full BXD panel can be used to identify and dissect the genetic mechanisms underlying these effects.

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

Impulsive behavior and impulsivity are heritable phenotypes that are strongly associated with risk for substance use disorders. Identifying the neurogenetic mechanisms that influence impulsivity may also reveal novel biological insights into addiction vulnerability. Our past studies using the BXD and Collaborative Cross (CC) recombinant inbred mouse panels have revealed that behavioral indicators of impulsivity measured in a reversal-learning task are heritable and are genetically correlated with aspects of intravenous cocaine self-administration. Genome-wide linkage studies in the BXD panel revealed a quantitative trait locus (QTL) on chromosome 10, but we expect to identify additional QTL by testing in a population with more genetic diversity. To this end, we turned to Diversity Outbred (DO) mice; 392 DO mice (156 males, 236 females) were phenotyped using the same reversal learning test utilized previously. Our primary indicator of impulsive responding, a measure that isolates the relative difficulty mice have with reaching performance criteria under reversal conditions, revealed a genome-wide significant QTL on chromosome 7 (max LOD score = 8.73, genome-wide corrected p<0.05). A measure of premature responding akin to that implemented in the 5-choice serial reaction time task yielded a suggestive QTL on chromosome 17 (max LOD score = 9.14, genome-wide corrected <0.1). Candidate genes were prioritized (2900076A07Rik, Wdr73 and Zscan2) based upon expression QTL data we collected in DO and CC mice and analyses using publicly available gene expression and phenotype databases. These findings may advance understanding of the genetics that drive impulsive behavior and enhance risk for substance use disorders.

A Locus on Chromosome 15 Contributes to Acute Ozone-induced Lung Injury in Collaborative Cross Mice.

Ozone (O3)-induced respiratory toxicity varies considerably within the human population and across inbred mouse strains, indicative of gene-environment interactions (GxE). Though previous studies have identified several quantitative trait loci (QTL) and candidate genes underlying responses to O3 exposure, precise mechanisms of susceptibility remain incompletely described. We sought to update our understanding of the genetic architecture of O3 responsiveness using the Collaborative Cross (CC) recombinant inbred mouse panel. We evaluated hallmark O3-induced inflammation and injury phenotypes in 56 CC strains after exposure to filtered air or 2 ppm O3, and performed focused genetic analysis of variation in lung injury, as reflected by protein in lung lavage fluid. Strain-dependent responses to O3 were clear, and QTL mapping revealed two novel loci on Chr (Chromosomes) 10 (peak, 26.2 Mb; 80% confidence interval [CI], 24.6-43.6 Mb) and 15 (peak, 47.1 Mb; 80% CI, 40.2-54.9 Mb), the latter surpassing the 95% significance threshold. At the Chr 15 locus, C57BL/6J and CAST/EiJ founder haplotypes were associated with higher lung injury responses compared with all other CC founder haplotypes. With further statistical analysis and a weight of evidence approach, we delimited the Chr 15 QTL to an ∼2 Mb region containing 21 genes (10 protein coding) and nominated three candidate genes, namely Oxr1, Rspo2, and Angpt1. Gene and protein expression data further supported Oxr1 and Angpt1 as priority candidate genes. In summary, we have shown that O3-induced lung injury is modulated by genetic variation, identified two high priority candidate genes, and demonstrated the value of the CC for detecting GxE.

Hybrid Rat Diversity Program (HRDP): A rat resource for mapping complex traits

Rats are a major model for studying complex disease mechanisms, behavioral phenotypes, environmental factors, and for drug development and discovery. Inbred rat strains control for genetic background and allow for repeated, reproducible molecular, cellular, and whole animal phenotyping. Genetic susceptibility to disease, sensitivity to environmental elements, and pharmacogenomics are critical components of the concepts of precision medicine. The Hybrid Rat Diversity Program (HRDP) is a resource that combines the power of genetic stability within strains, power for genomic mapping strategies, and strength in an animal model that mirrors many human disease traits. The HRDP was designed to include 96 inbred rat strains with genomic and physiological diversity to maximize power to detect specific genetic loci associated with complex traits while maximizing the genetic diversity among strains. The 96 strain panel consists of 33 genetically diverse inbred strains and two panels of recombinant inbred panels: FEXL/LEXF (33 strains, Japan) and HXB/BXH (30 strains, Czech Republic). Embryo resuscitation and breeding is well underway at the Medical College of Wisconsin (MCW) with 55 strains available. Whole genome sequencing for 54 of the 96 strains is complete, having been generated on am Illumina NovaSeq S4 to achieve 30X coverage. An additional 14 related substrains have been sequenced. Liver and brain transcriptome analysis is underway through PhenoGen (http://phenogen.ucdenver.edu) at the University of Colorado. Genomic, phenotype, and strain information will be made available through the Hybrid Rat Diversity Panel portal at the Rat Genome Database (http://rgd.mcw.edu). The HRDP will provide a genetically stable population of rat strains with fully sequenced genomes, transcriptomes for brain and liver, and general phenotypic characterization to be used for systems genetic studies and fine mapping of complex traits.

Beyond Genes: Inclusion of Alternative Splicing and Alternative Polyadenylation to Assess the Genetic Architecture of Predisposition to Voluntary Alcohol Consumption in Brain of the HXB/BXH Recombinant Inbred Rat Panel

Post transcriptional modifications of RNA are powerful mechanisms by which eukaryotes expand their genetic diversity. For instance, researchers estimate that most transcripts in humans undergo alternative splicing and alternative polyadenylation. These splicing events produce distinct RNA molecules, which in turn yield distinct protein isoforms and/or influence RNA stability, translation, nuclear export, and RNA/protein cellular localization. Due to their pervasiveness and impact, we hypothesized that alternative splicing and alternative polyadenylation in brain can contribute to a predisposition for voluntary alcohol consumption. Using the HXB/BXH recombinant inbred rat panel (a subset of the Hybrid Rat Diversity Panel), we generated over one terabyte of brain RNA sequencing data (total RNA) and identified novel splice variants (via StringTie) and alternative polyadenylation sites (via aptardi) to determine the transcriptional landscape in the brains of these animals. After establishing an analysis pipeline to ascertain high quality transcripts, we quantitated transcripts and integrated genotype data to identify candidate transcript coexpression networks and individual candidate transcripts associated with predisposition to voluntary alcohol consumption in the two-bottle choice paradigm. For genes that were previously associated with this trait (e.g., Lrap, Ift81, and P2rx4) (Saba et al., Febs. J., 282, 3556–3578, Saba et al., Genes. Brain. Behav., 20, e12698), we were able to distinguish between transcript variants to provide further information about the specific isoforms related to the trait. We also identified additional candidate transcripts associated with the trait of voluntary alcohol consumption (i.e., isoforms of Mapkapk5, Aldh1a7, and Map3k7). Consistent with our previous work, our results indicate that transcripts and networks related to inflammation and the immune system in brain can be linked to voluntary alcohol consumption. Overall, we have established a pipeline for including the quantitation of alternative splicing and alternative polyadenylation variants in the transcriptome in the analysis of the relationship between the transcriptome and complex traits.

Working memory and pattern separation in founder strains of the BXD recombinant inbred mouse panel

Working memory and pattern separation are fundamental cognitive abilities which, when impaired, significantly diminish quality of life. Discovering genetic mechanisms underlying innate and disease-induced variation in these cognitive abilities is a critical step towards treatments for common and devastating neurodegenerative conditions such as Alzheimer's disease. In this regard, the trial-unique nonmatching-to-location assay (TUNL) is a touchscreen operant conditioning procedure allowing simultaneous quantification of working memory and pattern separation in mice and rats. In the present study, we used the TUNL assay to quantify these cognitive abilities in C57BL/6J and DBA/2J mice. These strains are the founders of the BXD recombinant inbred mouse panel which enables discovery of genetic mechanisms underlying phenotypic variation. TUNL testing revealed that pattern separation was significantly influenced by mouse strain, whereas working memory was not. Moreover, horizontal distance and vertical distance between choice-phase stimuli had dissociable effects on TUNL performance. These findings provide novel data on mouse strain differences in pattern separation and support previous findings of equivalent working memory performance in C57BL/6J and DBA/2J mice. Although working memory of the BXD founder strains was equivalent in this study, working memory of BXD strains may be divergent because of transgressive segregation. Collectively, data presented here indicate that pattern separation is heritable in the mouse and that the BXD panel can be used to identify mechanisms underlying variation in pattern separation.

Incorporating genetic diversity to improve alignment of mouse models to human Alzheimer’s disease

Previous work by our labs, and others, reveal the benefits of incorporating diverse mouse strains to better model human Alzheimer's disease (AD). For instance, mouse models of AD on genetically distinct wild-derived mouse strains demonstrated robust differences in immune response to amyloid and neurodegeneration, better representing the phenotypic spectrum of AD than previous models. To further investigate the importance of genetic diversity, as part of MODEL-AD, we generated AD-relevant mouse models on Collaborative Cross (CC) lines background, a recombinant inbred mouse panel created from eight highly diverse founder strains.C57BL/6J (B6J) mice homozygous for humanized APOE4 allele and carrying mutant APP and PS1 alleles (APPswe , PS1de9 ) were crossed to five CC strains that were selected based on variation in AD-relevant genes. Cohorts of male and female B6JCCnF1.APOE4.APP/PS1 mice and controls were aged to 8 months (a mid-point for amyloid deposition in B6.APP/PS1 mice) and brain hemispheres processed for RNA-Seq and neuropathological assessment. Expectation Maximization for Allele Specific Expression (EMASE) tool was used to quantify transcriptome-wide expression at allelic level. Differential expression and KEGG pathway enrichment analysis were performed to determine altered molecular processes in CC strains. Strain specific transcriptional signatures were mapped to co-expression gene modules of human Late-Onset AD (LOAD) from the AMP-AD consortium. Neuropathological assessment included characterization of plaque deposition, Tau pathology, glial activation, neurodegeneration and cerebrovascular health.RNA-Seq analysis revealed up-regulation of neuroinflammation related pathways (lysosome, complement and coagulation cascades) in all CC strains as an effect of mutant APP and PS1 transgenes in presence of APOE4 allele. On the other hand, we observed significant CC strain and sex specific variation in immune (Fc gamma R-mediated phagocytosis) and metabolic (glycosphingolipid biosynthesis) response to amyloid pathogenesis. Alignment with human LOAD signatures revealed high strain and sex specific variation in correlation with synaptic signaling, cell cycle and DNA repair related gene modules. Overall, novel AD mouse models that incorporated CC lines demonstrated increased alignment to human AD-like transcriptomic signatures compared to B6J.This work builds on previous work highlighting the strengths of including multiple diverse mouse strains to better model complex neurodegenerative disorders such as AD and related dementias.

Strain and sex dependent effects of isolation housing relative to environmental enrichment on operant sensation seeking in mice

Sensation seeking is a multidimensional phenotype that predicts the development of drug addiction in humans and addiction-like drug seeking in rodents. Several lines of evidence suggest that chronic stress increases sensation seeking and addiction-like drug seeking through common genetic mechanisms. Discovery and characterization of these mechanisms would reveal how chronic stress interacts with the genome to influence sensation seeking and how drugs of abuse hijack these fundamental reward mechanisms to drive addiction. To this end, we tested the hypothesis that chronic isolation housing stress (relative to environmental enrichment) influences operant sensation seeking as a function of strain, sex, or their interaction. To determine if the BXD recombinant inbred panel could be used to identify genetic and epigenetic mechanisms underlying any identified gene-by-environment interactions, we used mice from the two BXD founder strains. Following 10 weeks of differential housing, we assessed operant sensation seeking using several reinforcement schedules. The primary finding from this study was that DBA/2J but not C57BL/6J mice were significantly vulnerable to an isolation-induced increase (relative to environmental enrichment) in sensation seeking during extinction when the sensory reward was no longer available; this effect was significantly more robust in females. These data reveal a previously unknown isolation-induced effect on extinction of operant sensation seeking that is sex-dependent, addiction-relevant, and that can be dissected using the BXD recombinant inbred panel.

Systems genetic analysis of nicotine withdrawal deficits in hippocampus-dependent learning.

Cognitive deficits, such as disrupted learning, are a major symptom of nicotine withdrawal. These deficits are heritable, yet their genetic basis is largely unknown. Our lab has developed a mouse model of nicotine withdrawal deficits in learning, using chronic nicotine exposure via osmotic minipumps and fear conditioning. Here, we utilized the BXD genetic reference panel to identify genetic variants underlying nicotine withdrawal deficits in learning. Male and female mice (n = 6-11 per sex per strain, 31 strains) received either chronic saline or nicotine (6.3 mg/kg per day for 12 days), and were then tested for hippocampus-dependent learning deficits using contextual fear conditioning. Quantitative trait locus (QTL) mapping analyses using GeneNetwork identified a significant QTL on Chromosome 4 (82.13 Mb, LRS = 20.03, p < 0.05). Publicly available hippocampal gene expression data were used to identify eight positional candidates (Snacpc3, Mysm1, Rps6, Plaa, Lurap1l, Slc24a2, Hacd4, Ptprd) that overlapped with our behavioral QTL and correlated with our behavioral data. Overall, this study demonstrates that genetic factors impact cognitive deficits during nicotine withdrawal in the BXD recombinant inbred panel and identifies candidate genes for future research.

Systems genetics analysis of the LXS recombinant inbred mouse strains:Genetic and molecular insights into acute ethanol tolerance.

We have been using the Inbred Long- and Short-Sleep mouse strains (ILS, ISS) and a recombinant inbred panel derived from them, the LXS, to investigate the genetic underpinnings of acute ethanol tolerance which is considered to be a risk factor for alcohol use disorders (AUDs). Here, we have used RNA-seq to examine the transcriptome of whole brain in 40 of the LXS strains 8 hours after a saline or ethanol "pretreatment" as in previous behavioral studies. Approximately 1/3 of the 14,184 expressed genes were significantly heritable and many were unique to the pretreatment. Several thousand cis- and trans-eQTLs were mapped; a portion of these also were unique to pretreatment. Ethanol pretreatment caused differential expression (DE) of 1,230 genes. Gene Ontology (GO) enrichment analysis suggested involvement in numerous biological processes including astrocyte differentiation, histone acetylation, mRNA splicing, and neuron projection development. Genetic correlation analysis identified hundreds of genes that were correlated to the behaviors. GO analysis indicated that these genes are involved in gene expression, chromosome organization, and protein transport, among others. The expression profiles of the DE genes and genes correlated to AFT in the ethanol pretreatment group (AFT-Et) were found to be similar to profiles of HDAC inhibitors. Hdac1, a cis-regulated gene that is located at the peak of a previously mapped QTL for AFT-Et, was correlated to 437 genes, most of which were also correlated to AFT-Et. GO analysis of these genes identified several enriched biological process terms including neuron-neuron synaptic transmission and potassium transport. In summary, the results suggest widespread genetic effects on gene expression, including effects that are pretreatment-specific. A number of candidate genes and biological functions were identified that could be mediating the behavioral responses. The most prominent of these was Hdac1 which may be regulating genes associated with glutamatergic signaling and potassium conductance.

Collaborative Cross mice reveal extreme epilepsy phenotypes and genetic loci for seizure susceptibility.

Animal studies remain essential for understanding mechanisms of epilepsy and identifying new therapeutic targets. However, existing animal models of epilepsy do not reflect the high level of genetic diversity found in the human population. The Collaborative Cross (CC) population is a genetically diverse recombinant inbred panel of mice. The CC offers large genotypic and phenotypic diversity, inbred strains with stable genomes that allow for repeated phenotypic measurements, and genomic tools including whole genome sequence to identify candidate genes and candidate variants. We evaluated multiple complex epileptic traits in a sampling of 35 CC inbred strains using the flurothyl-induced seizure and kindling paradigm. We created an F2 population of 297 mice with extreme seizure susceptibility and performed quantitative trait loci (QTL) mapping to identify genomic regions associated with seizure sensitivity. We used quantitative RNA sequencing from CC hippocampal tissue to identify candidate genes and whole genome sequence to identify genetic variants likely affecting gene expression. We identified new mouse models with extreme seizure susceptibility, seizure propagation, epileptogenesis, and SUDEP (sudden unexpected death in epilepsy). We performed QTL mapping and identified one known and seven novel loci associated with seizure sensitivity. We combined whole genome sequencing and hippocampal gene expression to pinpoint biologically plausible candidate genes (eg, Gabra2) and variants associated with seizure sensitivity. New mouse models of epilepsy are needed to better understand the complex genetic architecture of seizures and to identify therapeutics. We performed a phenotypic screen utilizing a novel genetic reference population of CC mice. The data we provide enable the identification of protective/risk genes and novel molecular mechanisms linked to complex seizure traits that are currently challenging to study and treat.

Diverse CD8 T cell responses to infection revealed by the collaborative cross

Abstract Enhanced host protection against re-infection requires generation of memory T cells of sufficient quantity and functional quality. Unlike well-studied inbred mice, T cell responses of diverse size and quality are generated following infection of humans and outbred mice. Thus, additional models are needed that more accurately reflect variation in immune outcomes in genetically diverse populations and to uncover underlying genetic causes. The Collaborative Cross (CC), a large recombinant inbred panel of mice, is an ideal model in this pursuit due to the high degree of genetic variation present and because it allows for assessment of genetic factors underlying unique phenotypes. The objectives of this study were to determine if the CC models diversity of immune outcomes seen in the human population, and if it could be used to identify genetic factors contributing to the generation of qualitatively and quantitatively distinct memory CD8 T cells. To this end, we analyzed the T cell response following acute LCMV Armstrong infection in 47 strains of CC mice. We found variability in resting immune cell composition and adaptive immune responses generated among CC strains in response to infection and reveal quantitative trait loci and candidate genes responsible for generation of CD62L+ memory CD8 T cells. Our results support the use of the CC to model diversity in immune responses observed in genetically diverse organisms and to uncover regulatory networks and host genetic factors underlying diverse immune outcomes following infection. This study and future studies utilizing the CC have the potential to improve translational efforts for the generation of protective immune responses against cancer and infections of global importance.

E2f8 and Dlg2 genes have independent effects on impaired insulin secretion associated with hyperglycaemia.

Reduced insulin secretion results in hyperglycaemia and diabetes involving a complex aetiology that is yet to be fully elucidated. Genetic susceptibility is a key factor in beta cell dysfunction and hyperglycaemia but the responsible genes have not been defined. The Collaborative Cross (CC) is a recombinant inbred mouse panel with diverse genetic backgrounds allowing the identification of complex trait genes that are relevant to human diseases. The aim of this study was to identify and characterise genes associated with hyperglycaemia. Using an unbiased genome-wide association study, we examined random blood glucose and insulin sensitivity in 53 genetically unique mouse strains from the CC population. The influences of hyperglycaemia susceptibility quantitative trait loci (QTLs) were investigated by examining glucose tolerance, insulin secretion, pancreatic histology and gene expression in the susceptible mice. Expression of candidate genes and their association with insulin secretion were examined in human islets. Mechanisms underlying reduced insulin secretion were studied in MIN6 cells using RNA interference. Wide variations in blood glucose levels and the related metabolic traits (insulin sensitivity and body weight) were observed in the CC population. We showed that elevated blood glucose in the CC strains was not due to insulin resistance nor obesity but resulted from reduced insulin secretion. This insulin secretory defect was demonstrated to be independent of abnormalities in islet morphology, beta cell mass and pancreatic insulin content. Gene mapping identified the E2f8 (p = 2.19 × 10-15) and Dlg2 loci (p = 3.83 × 10-8) on chromosome 7 to be significantly associated with hyperglycaemia susceptibility. Fine mapping the implicated regions using congenic mice demonstrated that these two loci have independent effects on insulin secretion in vivo. Significantly, our results revealed that increased E2F8 and DLG2 gene expression are correlated with enhanced insulin secretory function in human islets. Furthermore, loss-of-function studies in MIN6 cells demonstrated that E2f8 is involved in insulin secretion through an ATP-sensitive K+ channel-dependent pathway, which leads to a 30% reduction in Abcc8 expression. Similarly, knockdown of Dlg2 gene expression resulted in impaired insulin secretion in response to glucose and non-glucose stimuli. Collectively, these findings suggest that E2F transcription factor 8 (E2F8) and discs large homologue 2 (DLG2) regulate insulin secretion. The CC resource enables the identification of E2f8 and Dlg2 as novel genes associated with hyperglycaemia due to reduced insulin secretion in pancreatic beta cells. Taken together, our results provide better understanding of the molecular control of insulin secretion and further support the use of the CC resource to identify novel genes relevant to human diseases.

Hybrid Rat Diversity Program (HRDP): A rat resource for mapping complex traits

Rats are a major model for studying complex disease mechanisms, behavioral phenotypes, environmental factors, and for drug development and discovery. Inbred rat strains control for genetic background and allow for repeated, reproducible molecular, cellular, and whole animal phenotyping. Genetic susceptibility to disease, sensitivity to environmental elements, and pharmacogenomics are critical components of the concepts of precision medicine. A resource that combines the power of genetic stability within strains, power for genomic mapping strategies, and strength in an animal model that mirrors many human disease traits is the goal of this project. Through the development of the Hybrid Rat Diversity Program (HRDP), a powerful panel of inbred rats with genomic, physiologic, and behavioral phenotype data is beginning to serve as a catalyst for research in many fields. When complete, the HRDP will consist of 96 inbred rat strains to maximize power to detect specific genetic loci associated with complex traits while maximizing the genetic diversity among strains. The 96 strain panel consists of 33 genetically diverse inbred strains and two panels of recombinant inbred panels: FEXL/LEXF (33 strains, Japan) and HXB/BXH (30 strains, Czech Republic). Embryo resuscitation and breeding is well underway at the Medical College of Wisconsin (MCW). Whole genome sequencing for 32 of the 96 strains is complete, having been generated on am Illumina NovaSeq S4 to achieve 30X coverage. Liver and brain transcriptome analysis is underway through PhenoGen ( http://phenogen.ucdenver.edu) at the University of Colorado. Genomic, phenotype, and strain information will be made available through the Hybrid Rat Diversity Panel portal at the Rat Genome Database ( http://rgd.mcw.edu). The HRDP will provide a genetically stable population of rat strains with fully sequenced genomes, transcriptomes for brain and liver, and general phenotypic characterization to be used for systems genetic studies and fine mapping of complex traits.Support or Funding InformationNational Institutes of Health, Office of the Director, R24OD024617

Diverse CD8 T Cell Responses to Viral Infection Revealed by the Collaborative Cross

SUMMARYEnhanced host protection against re-infection requires generation of memory T cells of sufficient quantity and functional quality. Unlike well-studied inbred mice, T cell responses of diverse size and quality are generated following infection of humans and outbred mice. Thus, additional models are needed that accurately reflect variation in immune outcomes in genetically diverse populations and to uncover underlying genetic causes. The Collaborative Cross (CC), a large recombinant inbred panel of mice, is an ideal model in this pursuit for the high degree of genetic variation present, because it allows for assessment of genetic factors underlying unique phenotypes. Here, we advance the utility of the CC as a tool to analyze the immune response to viral infection. We describe variability in resting immune cell composition and adaptive immune responses generated among CC strains following systemic virus infection and reveal quantitative trait loci responsible for generation of CD62L+ memory CD8 T cells.

Stimulus Complexity and Mouse Strain Drive Escalation of Operant Sensation Seeking Within and Across Sessions in C57BL/6J and DBA/2J Mice.

Sensation seeking is a heritable trait that is genetically correlated with substance use; the shared genetic mechanisms underlying these traits are largely unknown. The relationship of sensation seeking and substance use has practical importance because discovering genes that drive sensation seeking can reveal genes driving substance use, and quantification of sensation seeking in mice is higher throughput and less technically challenging than quantification of volitional drug use. In order to fully understand the genetic mechanisms driving sensation seeking, it is critical to first understand the nongenetic factors driving sensation seeking. In the present study, we used the operant sensation seeking paradigm to assess the effects of stimulus complexity on sensation seeking in C57BL/6J and DBA/2J mice. These strains are the founders of the BXD recombinant inbred mouse panel which enables the discovery of genes driving phenotypic variation. This study led to four principal conclusions. First, all sensory stimuli used in the study, regardless of complexity or number of stimulus modalities, served as reinforcers for C57BL/6J and DBA/2J mice. Second, for both C57BL/6J and DBA/2J mice, sensation seeking for a high complexity sensory stimulus was significantly greater than sensation seeking for a low complexity sensory stimulus. Third, for both C57BL/6J and DBA/2J mice, sensation seeking escalated significantly within-session when a multimodal sensory stimulus of medium or high complexity was used but not when a unimodal sensory stimulus of low complexity was used. Finally, both the magnitude of sensation seeking and the magnitude of within-session escalation of sensation seeking were significantly greater in mice from the DBA/2J strain relative to mice from the C57BL/6J strain. Collectively, these findings indicate that stimulus complexity and genetic background drive escalation of operant sensation seeking within and across sessions, and that the BXD recombinant inbred mouse panel can be used to discover the genetic mechanisms underlying these phenomena.

A Cross-Species Systems Genetics Analysis Links APBB1IP as a Candidate for Schizophrenia and Prepulse Inhibition.

Background: Prepulse inhibition (PPI) of the startle response is a highly conserved form of sensorimotor gating, disruption of which is found in schizophrenia patients and their unaffected first-degree relatives. PPI can be measured in many species, and shows considerable phenotypic variation between and within rodent models. This makes PPI a useful endophenotype. Genome-wide association studies (GWAS) have been carried out to identify genetic variants underlying schizophrenia, and these suggest that schizophrenia is highly polygenic. GWAS have been unable to account for the high heritability of schizophrenia seen in family studies, partly because of the low power of GWAS due to multiple comparisons. By contrast, complementary mouse model linkage studies often have high statistical power to detect variants for behavioral traits but lower resolution, producing loci that include tens or hundreds of genes. To capitalize on the advantages of both GWAS and genetic mouse models, our study uses a cross-species approach to identify novel genes associated with PPI regulation, which thus may contribute to the PPI deficits seen in schizophrenia.Results: Using experimental data from the recombinant inbred (RI) mouse panel BXD, we identified two significant loci affecting PPI. These genomic regions contain genetic variants which influence PPI in mice and are therefore candidates that may be influencing aspects of schizophrenia in humans. We next investigated these regions in whole-genome data from the Psychiatric Genomics Consortium (PGC) schizophrenia GWAS and identify one novel candidate gene (ABPP1IP) that was significantly associated with PPI in mice and risk of schizophrenia in humans. A systems genetics approach demonstrates that APBB1IP coexpresses with several other genes related to schizophrenia in several brain regions. Gene coexpression and enrichment analysis shows clear links between APBB1IP and the immune system.Conclusion: The combination of human GWAS and mouse quantitative trait loci (QTL) from some of the largest study systems available has enabled us to identify a novel gene, APBB1IP, which influences schizophrenia in humans and PPI in mice.

Insight into genetic regulation of miRNA in mouse brain

Backgroundmicro RNA (miRNA) are important regulators of gene expression and may influence phenotypes and disease traits. The connection between genetics and miRNA expression can be determined through expression quantitative loci (eQTL) analysis, which has been extensively used in a variety of tissues, and in both human and model organisms. miRNA play an important role in brain-related diseases, but eQTL studies of miRNA in brain tissue are limited. We aim to catalog miRNA eQTL in brain tissue using miRNA expression measured on a recombinant inbred mouse panel. Because samples were collected without any intervention or treatment (naïve), the panel allows characterization of genetic influences on miRNAs’ expression levels.We used brain RNA expression levels of 881 miRNA and 1416 genomic locations to identify miRNA eQTL. To address multiple testing, we employed permutation p-values and subsequent zero permutation p-value correction. We also investigated the underlying biology of miRNA regulation using additional analyses, including hotspot analysis to search for regions controlling multiple miRNAs, and Bayesian network analysis to identify scenarios where a miRNA mediates the association between genotype and mRNA expression. We used addiction related phenotypes to illustrate the utility of our results.ResultsThirty-eight miRNA eQTL were identified after appropriate multiple testing corrections. Ten of these miRNAs had target genes enriched for brain-related pathways and mapped to four miRNA eQTL hotspots. Bayesian network analysis revealed four biological networks relating genetic variation, miRNA expression and gene expression.ConclusionsOur extensive evaluation of miRNA eQTL provides valuable insight into the role of miRNA regulation in brain tissue. Our miRNA eQTL analysis and extended statistical exploration identifies miRNA candidates in brain for future study.