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Environmental bisphenol compounds like bisphenol F (BPF) are endocrine disrupting chemicals (EDC) affecting adipose and classical endocrine systems. Genetic factors that influence EDC exposure outcomes are poorly understood and are unaccounted variables that may contribute to the large range of reported outcomes in the human population. We previously demonstrated that BPF exposure increased body growth and adiposity in male N/NIH Heterogeneous Stock (HS) rats, a genetically heterogeneous outbred population. We hypothesize that the founder strains of the HS rat exhibit EDC effects that were strain- and sex-dependent. Weanling littermate pairs of male and female ACI, BN, BUF, F344, M520, and WKY rats randomly received either vehicle (0.1% EtOH) or 1.125 mg BPF/L in 0.1% EtOH for ten weeks in drinking water. Body weight and fluid intake were measured weekly, metabolic parameters were assessed, and blood and tissues were collected. BPF increased thyroid weight in ACI males, thymus and kidney weight in BUF females, adrenal weight in WKY males, and possibly increased pituitary weight in BN males. BUF females also developed a disruption in activity and metabolic rate with BPF exposure. These sex- and strain-specific exposure outcomes illustrate that HS rat founders possess diverse bisphenol-exposure risk alleles and suggests that BPF exposure may intensify inherent organ system dysfunction existing in the HS rat founders. We propose that the HS rat will be an invaluable model for dissecting gene EDC interactions on health.

Rare diseases individually affect relatively few people, but as a group they impact considerable numbers of people. The Rat Genome Database (https://rgd.mcw.edu) is a knowledgebase that offers resources for rare disease research. This includes disease definitions, genes, quantitative trail loci (QTLs), genetic variants, annotations to published literature, links to external resources, and more. One important resource is identifying relevant cell lines and rat strains that serve as models for disease research. Diseases, genes, and strains have report pages with consolidated data, and links to analysis tools. Utilizing these globally accessible resources for rare disease research, potentiating discovery of mechanisms and new treatments, can point researchers toward solutions to alleviate the suffering of those afflicted with these diseases.

The Rat Genome Database (RGD, https://rgd.mcw.edu) has evolved from simply a resource for rat genetic markers, maps, and genes, by adding multiple genomic data types and extensive disease and phenotype annotations and developing tools to effectively mine, analyze, and visualize the available data, to empower investigators in their hypothesis-driven research. Leveraging its robust and flexible infrastructure, RGD has added data for human and eight other model organisms (mouse, 13-lined ground squirrel, chinchilla, naked mole-rat, dog, pig, African green monkey/vervet, and bonobo) besides rat to enhance its translational aspect. This article presents an overview of the database with the most recent additions to RGD’s genome, variant, and quantitative phenotype data. We also briefly introduce Virtual Comparative Map (VCMap), an updated tool that explores synteny between species as an improvement to RGD’s suite of tools, followed by a discussion regarding the refinements to the existing PhenoMiner tool that assists researchers in finding and comparing quantitative data across rat strains. Collectively, RGD focuses on providing a continuously improving, consistent, and high-quality data resource for researchers while advancing data reproducibility and fulfilling Findable, Accessible, Interoperable, and Reusable (FAIR) data principles.

BackgroundWe have generated a rat model similar to the Four Core Genotypes mouse model, allowing comparison of XX and XY rats with the same type of gonad. The model detects novel sex chromosome effects (XX vs. XY) that contribute to sex differences in any rat phenotype.MethodsXY rats were produced with an autosomal transgene ofSry, the testis-determining factor gene, which were fathers of XX and XY progeny with testes. In other rats, CRISPR-Cas9 technology was used to remove Y chromosome factors that initiate testis differentiation, producing fertile XY gonadal females that have XX and XY progeny with ovaries. These groups can be compared to detect sex differences caused by sex chromosome complement (XX vs. XY) and/or by gonadal hormones (rats with testes vs. ovaries).ResultsWe have measured numerous phenotypes to characterize this model, including gonadal histology, breeding performance, anogenital distance, levels of reproductive hormones, body and organ weights, and central nervous system sexual dimorphisms. Serum testosterone levels were comparable in adult XX and XY gonadal males. Numerous phenotypes previously found to be sexually differentiated by the action of gonadal hormones were found to be similar in XX and XY rats with the same type of gonad, suggesting that XX and XY rats with the same type of gonad have comparable levels of gonadal hormones at various stages of development.ConclusionThe results establish a powerful new model to discriminate sex chromosome and gonadal hormone effects that cause sexual differences in rat physiology and disease.

The COVID-19 pandemic stemmed a parallel upsurge in the scientific literature about SARS-CoV-2 infection and its health burden. The Rat Genome Database (RGD) created a COVID-19 Disease Portal to leverage information from the scientific literature. In the COVID-19 Portal, gene-disease associations are established by manual curation of PubMed literature. The portal contains data for nine ontologies related to COVID-19, an embedded enrichment analysis tool, as well as links to a toolkit. Using these information and tools, we performed analyses on the curated COVID-19 disease genes. As expected, Disease Ontology enrichment analysis showed that the COVID-19 gene set is highly enriched with coronavirus infectious disease and related diseases. However, other less related diseases were also highly enriched, such as liver and rheumatic diseases. Using the comparison heatmap tool, we found nearly 60 percent of the COVID-19 genes were associated with nervous system disease and 40 percent were associated with gastrointestinal disease. Our analysis confirms the role of the immune system in COVID-19 pathogenesis as shown by substantial enrichment of immune system related Gene Ontology terms. The information in RGD’s COVID-19 disease portal can generate new hypotheses to potentiate novel therapies and prevention of acute and long-term complications of COVID-19.

Complex phenotypes such as blood pressure regulation involve multiple organ systems, tissues, and specific cell types. Transgenic rodent models with the expression of fluorescent proteins under the control of tissue- and cell-type-specific promoters are powerful tools for identifying and isolating specific cells but have been primarily limited to mice. Our long-term goal is to establish a Cre-driver rat resource for conditional and physiologically predictive rat models of human diseases. We developed two Cre-driver transgenic strains of rats (SS/PodocinCreERT2 and SS/VECadCre) capable of activating a second Cre-sensitive tdTomato reporter transgene integrated into the rat Rosa26 locus in the Dahl salt-sensitive hypertension model background. Cells with reporter activation were identified under fluorescence microscopy or harvested using fluorescence-activated cell sorting (FACS). A tamoxifen-inducible Cre transgene under the control of the Nphs2 (podocin) gene promoter was used to harvest fluorescently labeled podocytes after tamoxifen injection. A constitutive Cre under the control of the Cdh5 (VE-Cadherin) gene promoter was used for the isolation of endothelial cells. Correspondingly, tens to hundreds of thousands of podocytes or endothelial cells were isolated from a single kidney or mesenteric artery. Isolated cells were enriched for specific marker genes and could be used in downstream assays including single-cell transcriptional profiling. These new Cre/reporter dual transgenic rat models provide a much-needed resource for the biomedical research community to study the role of these cell types in a broad range of health and disease modeling studies.

Outcomes following cardiac injury are influenced by lifestyle and environment, as well as genetic background. Our interest lies in the latter, where very few genes have successfully been linked to heart failure. Contributors to this low success rate include high genetic diversity of patient populations, inability to control for non-genetic factors, and cellular complexities that contribute to outcomes. We propose utilizing the Hybrid Rat Diversity Panel (HRDP) to assess high throughput surrogate phenotypes, which can be predictive of outcomes after injury when measured in the basal state. This approach can offer a solution to the limitations of association studies for complex diseases like heart failure. Our surrogate phenotype of interest is cardiomyocyte ploidy, where high frequency of diploid cardiomyocytes is predictive of ability to regenerate and functional recovery. Conversely, an increased proportion of polyploid (≥8N) cardiomyocytes is associated with adverse ventricular remodeling and dilated cardiomyopathy. Having analyzed 50 of the 96 strains, we see that frequency of the MNDCMs varies from 1.3-20.3% across strains, while frequency of the highly polyploid CMs (≥8N) varies from 0.8-20.9%. Four strains have been selected for myocardial infarction (MI) studies, including M520 (17.4%) and F344 (20.3%) with high MNDCM content and GK (2.8%) and HXB23 (1.8%) with low MNDCM content. Following MI, both M520 and F344 rats exhibit a higher percentage of BrdU+ cardiomyocytes in the border zone region compared to HXB23 rats, supporting the hypothesis that strains with high MNDCM frequency would be better at mounting a proliferative response. This also translated to a greater functional improvement and smaller scars in M520 rats, but not in F344. Additionally, we looked at the size of cardiomyocytes following MI and found that HXB23 rats (20.9% ≥8N CMs) have an increased cross-sectional area in comparison to the other strains, which may be indicative of concentric hypertrophy in response to injury. The HRDP could serve as a powerful tool for mapping traits related to outcomes following MI, allowing us to identify potential gene candidates that may be interrogated further for future use in precision medicine-based therapies. Mapping studies are ongoing.

Substance use disorder (SUD) is associated with a cluster of cognitive disturbances that engender vulnerability to ongoing drug seeking and relapse. Two of these endophenotypes—risky decision-making and impulsivity—are amplified in individuals with substance use disorder and are augmented by repeated exposure to illicit drugs. Identifying genetic factors underlying variability in these traits is critical for early identification, prevention, and treatment of SUD-vulnerable individuals. Here, we compared risky decision-making and different facets of impulsivity between two fully inbred substrains of Lewis rats—LEW/NCrl and LEW/NHsd. We performed whole genome sequencing of both substrain to identify almost all relevant variants. We observed substantial differences in risky decision-making and impulsive behaviors. Relative to LEW/HHsd, the LEW/NCrl substrain accepts higher risk options in a decision-making task and higher rates of premature responses in the differential reinforcement of low rates of responding (DRL) task. These phenotypic differences were more pronounced in females than males. We defined a total of ∼9,000 polymorphisms between these substrains at 40X whole genome short-read coverage. Roughly half of variants are located within a single 1.5 Mb region of chromosome 8, but none impact protein-coding regions. In contrast, other variants are widely distributed, and of these 38 are predicted to cause protein-coding variants. In conclusion, Lewis rat substrains differ significantly in risk-taking and impulsivity and only a small number of easily mapped variants are likely to be causal. Sequencing combined with a reduced complexity cross (RCC) should enable identification of one or more variants underlying multiple complex addiction-relevant traits.

Hypertension (HTN) is a complex disease influenced by heritable genetic elements and environmental interactions. Dietary salt is among the most influential modifiable factors contributing to increased blood pressure (BP). It is well established that men and women develop BP impairment in different patterns and a recent emphasis has been placed on identifying mechanisms leading to the differences observed between the sexes in HTN development. The current work reported here builds on an extensive genetic mapping experiment that sought to identify genetic determinants of salt-sensitive (SS) HTN using the Dahl SS rat. BTG antiproliferation factor 2 ( Btg2) was previously identified by our group as a candidate gene contributing to SS HTN in female rats. In the current study, Btg2 was mutated using transcription activator-like effector nuclease (TALEN)-targeted gene disruption on the SSBN congenic rat background. The Btg2 mutated rats exhibited impaired BP and proteinuria responses to a high-salt diet compared with wild-type rats. Differences in body weight, mutant pup viability, skeletal morphology, and adult nephron density suggest a potential role for Btg2 in developmental signaling pathways. Subsequent cell cycle gene expression assessment provides several additional signaling pathways that Btg2 may function through during salt handling in the kidney. The expression analysis also identified several potential upstream targets that can be explored to further isolate therapeutic approaches for SS HTN.

The move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tissues that are difficult to reach. Here we discuss the consortium’s plans to develop and benchmark approaches to induce and measure genome modifications, and to define downstream functional consequences of genome editing within human cells. Central to this effort is a rigorous and innovative approach that requires validation of the technology through third-party testing in small and large animals. New genome editors, delivery technologies and methods for tracking edited cells in vivo, as well as newly developed animal models and human biological systems, will be assembled—along with validated datasets—into an SCGE Toolkit, which will be disseminated widely to the biomedical research community. We visualize this toolkit—and the knowledge generated by its applications—as a means to accelerate the clinical development of new therapies for a wide range of conditions., This Perspective discusses how the Somatic Cell Genome Editing Consortium aims to accelerate the implementation of safe and effective genome-editing therapies in the clinic.

Genomic sequence and gene expression association studies in animals and humans have identified genes that may be integral in the pathogenesis of various diseases. CD14 (cluster of differentiation 14)—a cell surface protein involved in innate immune system activation—is one such gene associated with cardiovascular and hypertensive disease. We previously showed that this gene is upregulated in renal macrophages of Dahl salt-sensitive animals fed a high-salt diet; here we test the hypothesis that CD14 contributes to the elevated pressure and renal injury observed in salt-sensitive hypertension. Using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat–associated 9), we created a targeted mutation in the CD14 gene on the Dahl SS (SS/JrHSDMcwi) background and validated the absence of CD14 peptides via mass spectrometry. Radiotelemetry was used to monitor blood pressure in wild-type and CD14 −/ − animals challenged with high salt and identified infiltrating renal immune cells via flow cytometry. Germline knockout of CD14 exacerbated salt-sensitive hypertension and renal injury in female animals but not males. CD14 −/− females demonstrated increased infiltrating macrophages but no difference in infiltrating lymphocytes. Transplant of CD14 +/+ or CD14 −/− bone marrow was used to isolate the effects of CD14 knockout to hematopoietic cells and confirmed that the differential phenotype observed was due to knockout of CD14 in hematopoietic cells. Ovariectomy was used to remove the influence of female sex hormones, which completely abrogated the effect of CD14 knockout. These studies provide a novel treatment target and evidence of a new dichotomy in immune activation between sexes within the context of hypertensive disease where CD14 regulates immune cell activation and renal injury.

Chromosomal substitution strategies provide a powerful tool to anonymously reveal the relationship between DNA sequence variants and a normal or disease phenotype of interest. Even in this age of CRISPR-Cas9 genome engineering, the knockdown or overexpression of a gene provides relevant information to our understanding of complex disease only when a close association of an allelic variant with the phenotype has first been established. Limitations of genetic linkage approaches led to the development of more efficient breeding strategies to substitute chromosomal segments from one animal strain into the genetic background of a different strain, enabling a direct comparison of the phenotypes of the strains with variant(s) that differ only at a defined locus. This substitution can be a whole chromosome (consomic), a part of a chromosome (congenic), or as small as only a single or several alleles (subcongenics). In contrast to complete knockout of a specific candidate gene of interest, which simply studies the effects of complete elimination of the gene, the substitution of naturally occurring variants can provide special insights into the functional actions of wild-type alleles. Strategies for production of these inbred strains are reviewed, and a number of examples are used to illustrate the utility of these model systems. Consomic/congenic strains provide a number of experimental advantages in the study of functions of genes and their variants, which are emphasized in this article, such as replication of experimental studies; determination of temporal relationships throughout a life; rigorously controlled experiments in which relations between genotype and phenotype can be tested with the confounding effects of heterogeneous genetic backgrounds, both targeted and multilayered; and "omic" studies performed at many levels of functionality, from molecules to organelles, cells to organs, and organs to organismal behavior across the life span. The application of chromosomal substitution strategies and development of consomic/congenic rat and mouse strains have greatly expanded our knowledge of genomic variants and their phenotypic relationship to physiological functions and to complex diseases such as hypertension and cancer. © 2020 American Physiological Society. Compr Physiol 10:365-388, 2020.

Measuring animal behavior in the context of experimental manipulation is critical for modeling, and understanding neuropsychiatric disease. Prepulse inhibition of the acoustic startle response (PPI) is a behavioral phenomenon studied extensively for this purpose, but the results of PPI studies are often inconsistent. As a result, the utility of this phenomenon remains uncertain. Here, we deconstruct the phenomenon of PPI and confirm several limitations of the methodology traditionally utilized to describe PPI, including that the underlying startle response has a non-Gaussian distribution, and that the traditional PPI metric changes with different stimuli. We then develop a novel model that reveals PPI to be a combination of the previously appreciated scaling of the startle response, as well as a scaling of sound processing. Using our model, we find no evidence for differences in PPI in a rat model of Fragile-X Syndrome (FXS) compared with wild-type controls. These results in the rat provide a reliable methodology that could be used to clarify inconsistent PPI results in mice and humans. In contrast, we find robust differences between wild-type male and female rats. Our model allows us to understand the nature of these differences, and we find that both the startle-scaling and sound-scaling components of PPI are a function of the baseline startle response. Males and females differ specifically in the startle-scaling, but not the sound-scaling, component of PPI. These findings establish a robust experimental and analytical approach that has the potential to provide a consistent biomarker of brain function.

Biological interpretation of a large amount of gene or protein data is complex. Ontology analysis tools are imperative in finding functional similarities through overrepresentation or enrichment of terms associated with the input gene or protein lists. However, most tools are limited by their ability to do ontology-specific and species-limited analyses. Furthermore, some enrichment tools are not updated frequently with recent information from databases, thus giving users inaccurate, outdated or uninformative data. Here, we present MOET or the Multi-Ontology Enrichment Tool (v.1 released in April 2019 and v.2 released in May 2021), an ontology analysis tool leveraging data that the Rat Genome Database (RGD) integrated from in-house expert curation and external databases including the National Center for Biotechnology Information (NCBI), Mouse Genome Informatics (MGI), The Kyoto Encyclopedia of Genes and Genomes (KEGG), The Gene Ontology Resource, UniProt-GOA, and others. Given a gene or protein list, MOET analysis identifies significantly overrepresented ontology terms using a hypergeometric test and provides nominal and Bonferroni corrected P-values and odds ratios for the overrepresented terms. The results are shown as a downloadable list of terms with and without Bonferroni correction, and a graph of the P-values and number of annotated genes for each term in the list. MOET can be accessed freely from https://rgd.mcw.edu/rgdweb/enrichment/start.html.

Model organism research is essential for discovering the mechanisms of human diseases by defining biologically meaningful gene to disease relationships. The Rat Genome Database (RGD, (https://rgd.mcw.edu)) is a cross-species knowledgebase and the premier online resource for rat genetic and physiologic data. This rich resource is enhanced by the inclusion and integration of comparative data for human and mouse, as well as other human disease models including chinchilla, dog, bonobo, pig, 13-lined ground squirrel, green monkey, and naked mole-rat. Functional information has been added to records via the assignment of annotations based on sequence similarity to human, rat, and mouse genes. RGD has also imported well-supported cross-species data from external resources. To enable use of these data, RGD has developed a robust infrastructure of standardized ontologies, data formats, and disease- and species-centric portals, complemented with a suite of innovative tools for discovery and analysis. Using examples of single-gene and polygenic human diseases, we illustrate how data from multiple species can help to identify or confirm a gene as involved in a disease and to identify model organisms that can be studied to understand the pathophysiology of a gene or pathway. The ultimate aim of this report is to demonstrate the utility of RGD not only as the core resource for the rat research community but also as a source of bioinformatic tools to support a wider audience, empowering the search for appropriate models for human afflictions.

We present a genome assembly from an individual male Rattus norvegicus (the Norway rat; Chordata; Mammalia; Rodentia; Muridae). The genome sequence is 2.44 gigabases in span. The majority of the assembly is scaffolded into 20 chromosomal pseudomolecules, with both X and Y sex chromosomes assembled. This genome assembly, mRatBN7.2, represents the new reference genome for R. norvegicus and has been adopted by the Genome Reference Consortium.

Breast cancer risk is 31% heritable, yet the majority of the underlying risk factors remain poorly defined. Here, we used F2-linkage analysis in a rat mammary tumor model to identify a novel 11.2 Mb modifier locus of tumor incidence and burden on rat chromosome 5 (chr5: 15.4 – 26.6 Mb). Genomic and RNA sequencing analysis identified four differentially expressed candidates: TMEM68, IMPAD1, SDCBP, and RBM12B. Analysis of the human syntenic candidate region revealed that SDCBP is in close proximity to a previously reported genetic risk locus for human breast cancer. Moreover, analysis of the candidate genes in The Cancer Genome Atlas (TCGA) revealed that they fall within the commonly amplified 8q12.1 and 8q22.1 regions in human breast cancer patients and are correlated with worse overall survival. Collectively, this study presents novel evidence suggesting that TMEM68, IMPAD1, SDCBP, and RBM12B are potential modifiers of human breast cancer risk and outcome.

To investigate T helper type 17 (Th17) cells in the setting of acute kidney injury, the gene encoding the master regulator of Th17 cell differentiation, that is, RAR-related orphan receptor-γ (RORγT), was mutated in Lewis rats using CRISPR/Cas9 technology. In response to 40 min of bilateral renal ischemia-reperfusion (I/R), RAR-related orphan receptor C ( Rorc)−/− rats were resistant to injury relative to wild-type Rorc+/+ rats. This protection was associated with inhibition of IL-17 expression and reduced infiltration of CD4+ cells, CD8+ cells, B cells, and macrophages. To evaluate the effect of Th17 cells on repair, ischemia was increased to 50 min in Rorc−/− rats. This maneuver equalized the initial level of injury in Rorc−/− and Rorc+/+ rats 1 to 2 days post-I/R based on serum creatinine values. However, Rorc−/− rats, but not Rorc+/+ rats, failed to successfully recover renal function and had high mortality by 4 days post-I/R. Histological assessment of kidney tubules showed evidence of repair by day 4 post-I/R in Rorc+/+ rats but persistent necrosis and elevated cell proliferation in Rorc−/− rats. Adoptive transfer of CD4+ cells from the spleen of Rorc+/+ rats or supplementation of exogenous rIL-17 by an osmotic minipump improved renal function and survival of Rorc−/− rats following 50 min of I/R. This was associated with a relative decrease in the number of M1-type macrophages and a relative increase in the percentage of T regulatory cells. Taken together, these data suggest that Th17 cells have both a deleterious and a beneficial role in kidney injury and recovery, contributing to early postischemic injury and inflammation but also possibly being critical in the resolution of inflammation during kidney repair.

Hypertension (HTN) is a complex disease influenced by heritable genetic elements and environmental interactions. Dietary salt is among the most influential modifiable factors contributing to increased blood pressure (BP). It is well established that men and women develop BP impairment in different patterns and a recent emphasis has been placed on identifying mechanisms leading to the differences observed between the sexes in HTN development. The current work reported here builds on an extensive genetic mapping experiment which sought to identify genetic determinants of salt sensitive (SS) HTN using the Dahl SS rat. BTG anti-proliferation factor 2 ( Btg2 ) was previously isolated by our group using congenic breeding experiments which identified it as a female causative SS HTN candidate gene. In the current study, Btg2 was mutated using TALEN targeted gene disruption on the SSBN congenic rat background. The Btg2 mutated rats exhibited impaired BP and proteinuria responses to a high salt diet compared to wild type littermates. Differences in body weight, mutant pup viability, skeletal morphology, and adult nephron density suggest a potential role for Btg2 in developmental signaling pathways. Subsequent cell cycle gene expression assessment provides several additional signaling pathways that Btg2 may function through during salt handling in the kidney. The expression analysis also indicates several upstream targets that can be explored to further isolate therapeutic approaches for SS HTN.

BackgroundThe commonly used laboratory rat,Rattus norvegicus, is unique in having multipleSrygene copies found on the Y chromosome, with different copies encoding amino acid variations that influence the resulting protein function. It is not clear whichSrygenes are expressed at the onset of testis differentiation or how their expression correlates with that of other genes in testis-determination pathways.MethodsHere, two independent E11–E14 developmental RNAseq datasets show that multipleSrygenes are expressed at E12–E13.ResultsThe identified copies expressed during testis initiation includeSry4A,Sry1, andSry3C, which are conserved in every strain ofRattus norvegicuswith genomes sequenced to date.ConclusionsThis work represents a first step in defining the complex environment of rat testis differentiation that can open the door for generating sex reversal model systems using embryo manipulation techniques that have been available in the mouse but not the rat.