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1.  Zinc-Finger Nuclease Knockout of Dual-Specificity Protein Phosphatase-5 Enhances the Myogenic Response and Autoregulation of Cerebral Blood Flow in FHH.1BN Rats 
PLoS ONE  2014;9(11):e112878.
We recently reported that the myogenic responses of the renal afferent arteriole (Af-Art) and middle cerebral artery (MCA) and autoregulation of renal and cerebral blood flow (RBF and CBF) were impaired in Fawn Hooded hypertensive (FHH) rats and were restored in a FHH.1BN congenic strain in which a small segment of chromosome 1 from the Brown Norway (BN) containing 15 genes including dual-specificity protein phosphatase-5 (Dusp5) were transferred into the FHH genetic background. We identified 4 single nucleotide polymorphisms in the Dusp5 gene in FHH as compared with BN rats, two of which altered CpG sites and another that caused a G155R mutation. To determine whether Dusp5 contributes to the impaired myogenic response in FHH rats, we created a Dusp5 knockout (KO) rat in the FHH.1BN genetic background using a zinc-finger nuclease that introduced an 11 bp frame-shift deletion and a premature stop codon at AA121. The expression of Dusp5 was decreased and the levels of its substrates, phosphorylated ERK1/2 (p-ERK1/2), were enhanced in the KO rats. The diameter of the MCA decreased to a greater extent in Dusp5 KO rats than in FHH.1BN and FHH rats when the perfusion pressure was increased from 40 to 140 mmHg. CBF increased markedly in FHH rats when MAP was increased from 100 to 160 mmHg, and CBF was better autoregulated in the Dusp5 KO and FHH.1BN rats. The expression of Dusp5 was higher at the mRNA level but not at the protein level and the levels of p-ERK1/2 and p-PKC were lower in cerebral microvessels and brain tissue isolated from FHH than in FHH.1BN rats. These results indicate that Dusp5 modulates myogenic reactivity in the cerebral circulation and support the view that a mutation in Dusp5 may enhance Dusp5 activity and contribute to the impaired myogenic response in FHH rats.
doi:10.1371/journal.pone.0112878
PMCID: PMC4232417  PMID: 25397684
2.  Disease pathways at the Rat Genome Database Pathway Portal: genes in context—a network approach to understanding the molecular mechanisms of disease 
Human Genomics  2014;8(1):17.
Background
Biological systems are exquisitely poised to respond and adjust to challenges, including damage. However, sustained damage can overcome the ability of the system to adjust and result in a disease phenotype, its underpinnings many times elusive. Unraveling the molecular mechanisms of systems biology, of how and why it falters, is essential for delineating the details of the path(s) leading to the diseased state and for designing strategies to revert its progression. An important aspect of this process is not only to define the function of a gene but to identify the context within which gene functions act. It is within the network, or pathway context, that the function of a gene fulfills its ultimate biological role. Resolving the extent to which defective function(s) affect the proceedings of pathway(s) and how altered pathways merge into overpowering the system’s defense machinery are key to understanding the molecular aspects of disease and envisioning ways to counteract it. A network-centric approach to diseases is increasingly being considered in current research. It also underlies the deployment of disease pathways at the Rat Genome Database Pathway Portal. The portal is presented with an emphasis on disease and altered pathways, associated drug pathways, pathway suites, and suite networks.
Results
The Pathway Portal at the Rat Genome Database (RGD) provides an ever-increasing collection of interactive pathway diagrams and associated annotations for metabolic, signaling, regulatory, and drug pathways, including disease and altered pathways. A disease pathway is viewed from the perspective of networks whose alterations are manifested in the affected phenotype. The Pathway Ontology (PW), built and maintained at RGD, facilitates the annotations of genes, the deployment of pathway diagrams, and provides an overall navigational tool. Pathways that revolve around a common concept and are globally connected are presented within pathway suites; a suite network combines two or more pathway suites.
Conclusions
The Pathway Portal is a rich resource that offers a range of pathway data and visualization, including disease pathways and related pathway suites. Viewing a disease pathway from the perspective of underlying altered pathways is an aid for dissecting the molecular mechanisms of disease.
doi:10.1186/s40246-014-0017-8
PMCID: PMC4191248  PMID: 25265995
Molecular pathway; Disease pathway; Altered pathway; Ontology; Systems biology
3.  Rat Chromosome 8 Confers Protection against Dyslipidemia Caused by a High Fat/Low Carbohydrate Diet 
Background/Aims
Recent studies have highlighted the importance of gene by diet interactions in contributing to risk factors of metabolic syndrome. We used a consomic rat panel, in which a chromosome of the Brown Norway (BN) strain is introgressed onto the background of the Dahl Salt-Sentitive (SS) strain, to test the hypothesis that these animals will be useful for dissecting gene by diet interactions involved in metabolic syndrome.
Methods
We placed the parental SS and BN strains on a low fat/high carbohydrate (LF) or high fat/low carbohydrate (HF) diet for 22 weeks and measured several indices of metabolic syndrome. We then investigated the effect of diet in eight consomic rat strains.
Results
We show that the HF diet resulted in significantly increased levels of fasting plasma cholesterol and triglycerides in the SS strain, with no effect in the BN. Both strains responded to the HF diet with slight increases in body weight. SSBN8 was the only consomic strain that resembled that of the BN, with low levels of fasting cholesterol and triglycerides even on the HF diet.
Conclusions
These results indicate that BN chromosome 8 harbors a gene or genes that confer protection against dyslipidemia caused by the HF diet.
doi:10.1159/000338848
PMCID: PMC4167734  PMID: 22722880
Metabolic Syndrome; Consomic Rat Strains; Gene by environment interactions; genetic mapping
4.  Female-specific hypertension loci on rat chromosome 13 
Hypertension  2013;62(3):10.1161/HYPERTENSIONAHA.113.01708.
A 3.7 Mb region of rat chromosome 13 (45.2–49.0 Mb) affects blood pressure (BP) in females only, indicating the presence of gender-specific BP loci in close proximity to the Renin locus. In the present study, we used a series of Dahl salt-sensitive/Mcwi (SS)-13 Brown Norway (BN) congenic rat strains to further resolve BP loci within this region. We identified 3 BP loci affecting female rats only, of which the 2 smaller loci (line9BP3 and line9BP4) were functionally characterized by sequence and expression analysis. Compared with SS, the presence of a 591 Kb region of BN chromosome 13 (line9BP3) significantly lowered BP by 21 mmHg on an 8% NaCl diet (153±7 vs 174±5 mmHg, P<0.001). Unexpectedly, the addition of 23 Kb of BN chromosome 13 (line9BP4) completely erased the female-specific BP protection on 8% NaCl diet, suggesting that BN hypertensive allele(s) reside in this region. The congenic interval of the protective line 9F strain contains 3 genes (Optc, Prelp, and Fmod) and the hypertensive line 9E contains 1 additional gene (Btg2). Sequence analysis of the 2 BP loci revealed a total of 282 intergenic variants, with no coding variants. Analysis of gene expression by RT-qPCR revealed strain- and gender-specific differences in Prelp, Fmod, and Btg2 expression, implicating these as novel candidate genes for female-specific hypertension.
doi:10.1161/HYPERTENSIONAHA.113.01708
PMCID: PMC3870583  PMID: 23817491
Hypertension; Genetics; Gender; Blood Pressure; Kidney
5.  Identification of a Novel Gene for Diabetic Traits in Rats, Mice, and Humans 
Genetics  2014;198(1):17-29.
The genetic basis of type 2 diabetes remains incompletely defined despite the use of multiple genetic strategies. Multiparental populations such as heterogeneous stocks (HS) facilitate gene discovery by allowing fine mapping to only a few megabases, significantly decreasing the number of potential candidate genes compared to traditional mapping strategies. In the present work, we employed expression and sequence analysis in HS rats (Rattus norvegicus) to identify Tpcn2 as a likely causal gene underlying a 3.1-Mb locus for glucose and insulin levels. Global gene expression analysis on liver identified Tpcn2 as the only gene in the region that is differentially expressed between HS rats with glucose intolerance and those with normal glucose regulation. Tpcn2 also maps as a cis-regulating expression QTL and is negatively correlated with fasting glucose levels. We used founder sequence to identify variants within this region and assessed association between 18 variants and diabetic traits by conducting a mixed-model analysis, accounting for the complex family structure of the HS. We found that two variants were significantly associated with fasting glucose levels, including a nonsynonymous coding variant within Tpcn2. Studies in Tpcn2 knockout mice demonstrated a significant decrease in fasting glucose levels and insulin response to a glucose challenge relative to those in wild-type mice. Finally, we identified variants within Tpcn2 that are associated with fasting insulin in humans. These studies indicate that Tpcn2 is a likely causal gene that may play a role in human diabetes and demonstrate the utility of multiparental populations for positionally cloning genes within complex loci.
doi:10.1534/genetics.114.162982
PMCID: PMC4174929  PMID: 25236446
Tpcn2; heterogeneous stock rats; expression QTL mapping; type 2 diabetes; glucose; insulin; Multiparent Advanced Generation Inter-Cross (MAGIC); multiparental populations; MPP; gene mapping
6.  Congenic mapping and sequence analysis of the Renin locus 
Hypertension  2013;61(4):850-856.
Renin was the first blood pressure (BP) quantitative trait locus (QTL) mapped by linkage analysis in the rat. Subsequent BP linkage and congenic studies capturing different portions of the renin region have returned conflicting results, suggesting that multiple interdependent BP loci may be residing in the chromosome 13 BP QTL that includes Renin. We used SS-13BN congenic strains to map 2 BP loci in the Renin region (chr13:45.2–49.0 Mb). We identified a 1.1 Mb protective Brown Norway (BN) region around Renin (chr13:46.1–47.2 Mb) that significantly decreased BP by 32 mmHg. The Renin protective BP locus was offset by an adjacent hypertensive locus (chr13:47.2–49.0 Mb) that significantly increased BP by 29 mmHg. Sequence analysis of the protective and hypertensive BP loci revealed 1,433 and 2,063 variants between Dahl salt-sensitive/Mcwi (SS) and BN rats, respectively. To further reduce the list of candidate variants, we re-genotyped an overlapping SS-13SR congenic strain (S/renrr) with a previously reported BP phenotype. Sequence comparison between SS, Dahl R (SR), and BN reduced the number of candidate variants in the 2 BP loci by 42% for further study. Combined with previous studies, these data suggest that at least 4 BP loci reside within the 30 cM chromosome 13 BP QTL that includes Renin.
doi:10.1161/HYPERTENSIONAHA.111.01008
PMCID: PMC3770301  PMID: 23460292
hypertension; genetic; congenic; Dahl salt-sensitive rat; Brown Norway
7.  The pathway ontology – updates and applications 
Background
The Pathway Ontology (PW) developed at the Rat Genome Database (RGD), covers all types of biological pathways, including altered and disease pathways and captures the relationships between them within the hierarchical structure of a directed acyclic graph. The ontology allows for the standardized annotation of rat, and of human and mouse genes to pathway terms. It also constitutes a vehicle for easy navigation between gene and ontology report pages, between reports and interactive pathway diagrams, between pathways directly connected within a diagram and between those that are globally related in pathway suites and suite networks. Surveys of the literature and the development of the Pathway and Disease Portals are important sources for the ongoing development of the ontology. User requests and mapping of pathways in other databases to terms in the ontology further contribute to increasing its content. Recently built automated pipelines use the mapped terms to make available the annotations generated by other groups.
Results
The two released pipelines – the Pathway Interaction Database (PID) Annotation Import Pipeline and the Kyoto Encyclopedia of Genes and Genomes (KEGG) Annotation Import Pipeline, make available over 7,400 and 31,000 pathway gene annotations, respectively. Building the PID pipeline lead to the addition of new terms within the signaling node, also augmented by the release of the RGD “Immune and Inflammatory Disease Portal” at that time. Building the KEGG pipeline lead to a substantial increase in the number of disease pathway terms, such as those within the ‘infectious disease pathway’ parent term category. The ‘drug pathway’ node has also seen increases in the number of terms as well as a restructuring of the node. Literature surveys, disease portal deployments and user requests have contributed and continue to contribute additional new terms across the ontology. Since first presented, the content of PW has increased by over 75%.
Conclusions
Ongoing development of the Pathway Ontology and the implementation of pipelines promote an enriched provision of pathway data. The ontology is freely available for download and use from the RGD ftp site at ftp://rgd.mcw.edu/pub/ontology/pathway/ or from the National Center for Biomedical Ontology (NCBO) BioPortal website at http://bioportal.bioontology.org/ontologies/PW.
doi:10.1186/2041-1480-5-7
PMCID: PMC3922094  PMID: 24499703
Biological pathway; Ontology; Pipeline; Pathway annotations; Pathway diagrams
8.  Increased Proliferative Cells in the Medullary Thick Ascending Limb of the Loop of Henle in the Dahl Salt-Sensitive Rat 
Hypertension  2012;61(1):208-215.
Studies of transcriptome profiles have provided new insights into mechanisms underlying the development of hypertension. Cell type heterogeneity in tissue samples, however, has been a significant hindrance in these studies. We performed a transcriptome analysis in medullary thick ascending limbs of the loop of Henle isolated from Dahl salt-sensitive rats. Genes differentially expressed between Dahl salt-sensitive rats and salt-insensitive consomic SS.13BN rats on either 0.4% or 7 days of 8% NaCl diet (n=4) were highly enriched for genes located on chromosome 13, the chromosome substituted in the SS.13BN rat. A pathway involving cell proliferation and cell cycle regulation was identified as one of the most highly ranked pathways based on differentially expressed genes and by a Bayesian model analysis. Immunofluorescent analysis indicated that just one week of a high salt diet resulted in a several fold increase in proliferative medullary thick ascending limb cells in both rat strains and that Dahl salt-sensitive rats exhibited significantly greater proportion of medullary thick ascending limb cells in a proliferative state than in SS.13BN rats (15.0% ± 1.4% vs. 10.1% ± 0.6%, n=7–9, P<0.05). The total number of cells per medullary thick ascending limb section analyzed was not different between the two strains. The study revealed alterations in regulatory pathways in Dahl salt-sensitive rats in tissues highly enriched for a single cell type, leading to the unexpected finding of a greater increase in the number of proliferative medullary thick ascending limb cells in Dahl salt-sensitive rats on a high-salt diet.
doi:10.1161/HYPERTENSIONAHA.112.199380
PMCID: PMC3760736  PMID: 23184381
Kidney; gene expression; physiological genomics; cell cycle; salt intake
9.  Identification of Hypertension Susceptibility Loci on Rat Chromosome 12 
Hypertension  2012;60(4):10.1161/HYPERTENSIONAHA.112.198200.
Previous studies have identified multiple blood pressure and renal disease quantitative trait loci located on rat chromosome 12. In the present study, we narrowed blood pressure loci using a series of overlapping SS-12BN congenic lines. We found that transferring 6.1Mb of SS chromosome 12 (13.4-19.5Mb) onto the consomic SS-12BN background significantly elevated blood pressure on 1% NaCl (146±6 vs. 127±1 mmHg, P<0.01) and 8% NaCl diets (178±7 vs. 144±2 mmHg, P<0.05). Compared with the SS-12BN consomic, these animals also had significantly elevated albumin (218±31 vs. 104±8 mg/day, P<0.01) and protein excretion (347±41 vs. 195±12 mg/day, P<0.01) on 1% NaCl diet. Elevated blood pressure, albuminuria, and proteinuria coincided with greater renal and cardiac damage, demonstrating that SS allele(s) within the 6.1Mb congenic interval are associated with strong cardiovascular disease phenotypes. Sequence analysis of the 6.1Mb congenic region revealed 12,675 single nucleotide polymorphisms between SS and BN. Of these polymorphisms, 295 lie within coding regions and 20 resulted in nonsynonymous changes in conserved genes, of which 5 were predicted to be potentially damaging to protein function. Syntenic regions in human chromosome 7 have also been identified in multiple linkage and association studies of cardiovascular disease, suggesting that genetic variants underlying cardiovascular phenotypes in this congenic strain can likely be translated to a better understanding of human hypertension.
doi:10.1161/HYPERTENSIONAHA.112.198200
PMCID: PMC3873777  PMID: 22868394
Hypertension; Genetic; Dahl Salt-Sensitive Rat; Brown Norway; Congenic
10.  Exploring Genetic, Genomic, and Phenotypic Data at the Rat Genome Database 
The laboratory rat, Rattus norvegicus, is an important model of human health and disease, and experimental findings in the rat have relevance to human physiology and disease. The Rat Genome Database (RGD, http://rgd.mcw.edu) is a model organism database that provides access to a wide variety of curated rat data including disease associations, phenotypes, pathways, molecular functions, biological processes and cellular components for genes, quantitative trait loci, and strains. We present an overview of the database followed by specific examples that can be used to gain experience in employing RGD to explore the wealth of functional data available for the rat.
doi:10.1002/0471250953.bi0114s40
PMCID: PMC3555435  PMID: 23255149
rat; database; quantitative trait locus; ontology; genomics; gene
11.  Rat Strain Ontology: structured controlled vocabulary designed to facilitate access to strain data at RGD 
Background
The Rat Genome Database (RGD) ( http://rgd.mcw.edu/) is the premier site for comprehensive data on the different strains of the laboratory rat (Rattus norvegicus). The strain data are collected from various publications, direct submissions from individual researchers, and rat providers worldwide. Rat strain, substrain designation and nomenclature follow the Guidelines for Nomenclature of Mouse and Rat Strains, instituted by the International Committee on Standardized Genetic Nomenclature for Mice. While symbols and names aid in identifying strains correctly, the flat nature of this information prohibits easy search and retrieval, as well as other data mining functions. In order to improve these functionalities, particularly in ontology-based tools, the Rat Strain Ontology (RS) was developed.
Results
The Rat Strain Ontology (RS) reflects the breeding history, parental background, and genetic manipulation of rat strains. This controlled vocabulary organizes strains by type: inbred, outbred, chromosome altered, congenic, mutant and so on. In addition, under the chromosome altered category, strains are organized by chromosome, and further by type of manipulations, such as mutant or congenic. This allows users to easily retrieve strains of interest with modifications in specific genomic regions. The ontology was developed using the Open Biological and Biomedical Ontology (OBO) file format, and is organized on the Directed Acyclic Graph (DAG) structure. Rat Strain Ontology IDs are included as part of the strain report (RS: ######).
Conclusions
As rat researchers are often unaware of the number of substrains or altered strains within a breeding line, this vocabulary now provides an easy way to retrieve all substrains and accompanying information. Its usefulness is particularly evident in tools such as the PhenoMiner at RGD, where users can now easily retrieve phenotype measurement data for related strains, strains with similar backgrounds or those with similar introgressed regions. This controlled vocabulary also allows better retrieval and filtering for QTLs and in genomic tools such as the GViewer.
The Rat Strain Ontology has been incorporated into the RGD Ontology Browser ( http://rgd.mcw.edu/rgdweb/ontology/view.html?acc_id=RS:0000457#s) and is available through the National Center for Biomedical Ontology ( http://bioportal.bioontology.org/ontologies/1150) or the RGD ftp site ( ftp://rgd.mcw.edu/pub/ontology/rat_strain/).
doi:10.1186/2041-1480-4-36
PMCID: PMC4177145  PMID: 24267899
Rat strains; Phylogeny; RGD; Rat genome database
12.  Analyzing complex traits with congenic strains 
Congenic strains continue to be a fundamental resource for dissecting the genetic basis of complex traits. Traditionally, genetic variants (QTLs) that account for phenotypic variation in a panel of congenic strains are sought first by comparing phenotypes for each strain to the host (reference) strain, and then by examining the results to identify a common chromosome segment that provides the best match between genotype and phenotype across the panel. However, this ‘‘common-segment’’ method has significant limitations, including the subjective nature of the genetic model and an inability to deal formally with strain phenotypes that do not fit the model. We propose an alternative that we call ‘‘sequential’’ analysis and that is based on a unique principle of QTL analysis where each strain, corresponding to a single genotype, is tested individually for QTL effects rather than testing the congenic panel collectively for common effects across heterogeneous backgrounds. A minimum spanning tree, based on principles of graph theory, is used to determine the optimal sequence of strain comparisons. For two traits in two panels of congenic strains in mice, we compared results for the sequential method with the common-segment method as well as with two standard methods of QTL analysis, namely, interval mapping and multiple linear regression. The general utility of the sequential method was demonstrated with analysis of five additional traits in congenic panels from mice and rats. Sequential analysis rigorously resolved phenotypic heterogeneity among strains in the congenic panels and found QTLs that other methods failed to detect.
doi:10.1007/s00335-010-9267-5
PMCID: PMC3805105  PMID: 20524000
13.  Exploring Concordance and Discordance for Return of Incidental Findings from Clinical Sequencing 
Purpose
To explore specific conditions and types of genetic variants that specialists in genetics recommend should be returned as incidental findings in clinical sequencing.
Methods
Sixteen specialists in clinical genetics and/or molecular medicine selected variants in 99 common conditions to return to the ordering physician if discovered incidentally through whole genome sequencing. For most conditions, the specialists independently considered 3 molecular scenarios for both adults and minor children: a known pathogenic mutation, a truncating variant presumed pathogenic (where other truncating variants were known to be pathogenic), or a missense variant predicted in silico to be pathogenic.
Results
On average, for adults and children respectively, each specialist selected 83.5 and 79.0 conditions or genes out of 99 in the known pathogenic mutation categories, 57.0 and 53.5 out of 72 in the truncating variant categories, and 33.4 and 29.7 out of 72 in the missense variant categories. Concordance in favor of disclosure within the adult/known pathogenic mutation category was 100% for 21 conditions or genes and 80% or higher for 64 conditions or genes.
Conclusion
Specialists were highly concordant for the return of findings in 64 conditions or genes if discovered incidentally during whole exome or whole genome sequencing.
doi:10.1038/gim.2012.21
PMCID: PMC3763716  PMID: 22422049
whole genome sequencing; incidental findings
14.  Genome Sequencing Reveals Loci under Artificial Selection that Underlie Disease Phenotypes in the Laboratory Rat 
Cell  2013;154(3):691-703.
Summary
Large numbers of inbred laboratory rat strains have been developed for a range of complex disease phenotypes. To gain insights into the evolutionary pressures underlying selection for these phenotypes, we sequenced the genomes of 27 rat strains, including 11 models of hypertension, diabetes, and insulin resistance, along with their respective control strains. Altogether, we identified more than 13 million single-nucleotide variants, indels, and structural variants across these rat strains. Analysis of strain-specific selective sweeps and gene clusters implicated genes and pathways involved in cation transport, angiotensin production, and regulators of oxidative stress in the development of cardiovascular disease phenotypes in rats. Many of the rat loci that we identified overlap with previously mapped loci for related traits in humans, indicating the presence of shared pathways underlying these phenotypes in rats and humans. These data represent a step change in resources available for evolutionary analysis of complex traits in disease models.
PaperClip
Graphical Abstract
Highlights
•Genomes of 27 rat strains were sequenced; >13 million sequence variants identified•Selective sweeps and coevolved gene clusters were detected in 11 disease models•Previously identified and new disease genes and pathways were identified•This is first evolutionary analysis of artificial selection for disease phenotypes
Evolution analysis of artificial selection for disease phenotypes, such as hypertension and diabetes, in 27 rat strains reveals disease-related variants and loci.
doi:10.1016/j.cell.2013.06.040
PMCID: PMC3732391  PMID: 23890820
15.  Identification of somatic mutations in non-small cell lung carcinomas using whole-exome sequencing 
Carcinogenesis  2012;33(7):1270-1276.
Lung cancer is the leading cause of cancer-related death, with non-small cell lung cancer (NSCLC) being the predominant form of the disease. Most lung cancer is caused by the accumulation of genomic alterations due to tobacco exposure. To uncover its mutational landscape, we performed whole-exome sequencing in 31 NSCLCs and their matched normal tissue samples. We identified both common and unique mutation spectra and pathway activation in lung adenocarcinomas and squamous cell carcinomas, two major histologies in NSCLC. In addition to identifying previously known lung cancer genes (TP53, KRAS, EGFR, CDKN2A and RB1), the analysis revealed many genes not previously implicated in this malignancy. Notably, a novel gene CSMD3 was identified as the second most frequently mutated gene (next to TP53) in lung cancer. We further demonstrated that loss of CSMD3 results in increased proliferation of airway epithelial cells. The study provides unprecedented insights into mutational processes, cellular pathways and gene networks associated with lung cancer. Of potential immediate clinical relevance, several highly mutated genes identified in our study are promising druggable targets in cancer therapy including ALK, CTNNA3, DCC, MLL3, PCDHIIX, PIK3C2B, PIK3CG and ROCK2.
doi:10.1093/carcin/bgs148
PMCID: PMC3499051  PMID: 22510280
16.  Analysis of disease-associated objects at the Rat Genome Database 
The Rat Genome Database (RGD) is the premier resource for genetic, genomic and phenotype data for the laboratory rat, Rattus norvegicus. In addition to organizing biological data from rats, the RGD team focuses on manual curation of gene–disease associations for rat, human and mouse. In this work, we have analyzed disease-associated strains, quantitative trait loci (QTL) and genes from rats. These disease objects form the basis for seven disease portals. Among disease portals, the cardiovascular disease and obesity/metabolic syndrome portals have the highest number of rat strains and QTL. These two portals share 398 rat QTL, and these shared QTL are highly concentrated on rat chromosomes 1 and 2. For disease-associated genes, we performed gene ontology (GO) enrichment analysis across portals using RatMine enrichment widgets. Fifteen GO terms, five from each GO aspect, were selected to profile enrichment patterns of each portal. Of the selected biological process (BP) terms, ‘regulation of programmed cell death’ was the top enriched term across all disease portals except in the obesity/metabolic syndrome portal where ‘lipid metabolic process’ was the most enriched term. ‘Cytosol’ and ‘nucleus’ were common cellular component (CC) annotations for disease genes, but only the cancer portal genes were highly enriched with ‘nucleus’ annotations. Similar enrichment patterns were observed in a parallel analysis using the DAVID functional annotation tool. The relationship between the preselected 15 GO terms and disease terms was examined reciprocally by retrieving rat genes annotated with these preselected terms. The individual GO term–annotated gene list showed enrichment in physiologically related diseases. For example, the ‘regulation of blood pressure’ genes were enriched with cardiovascular disease annotations, and the ‘lipid metabolic process’ genes with obesity annotations. Furthermore, we were able to enhance enrichment of neurological diseases by combining ‘G-protein coupled receptor binding’ annotated genes with ‘protein kinase binding’ annotated genes.
Database URL: http://rgd.mcw.edu
doi:10.1093/database/bat046
PMCID: PMC3689439  PMID: 23794737
17.  Characterization of human plasma-derived exosomal RNAs by deep sequencing 
BMC Genomics  2013;14:319.
Background
Exosomes, endosome-derived membrane microvesicles, contain specific RNA transcripts that are thought to be involved in cell-cell communication. These RNA transcripts have great potential as disease biomarkers. To characterize exosomal RNA profiles systemically, we performed RNA sequencing analysis using three human plasma samples and evaluated the efficacies of small RNA library preparation protocols from three manufacturers. In all we evaluated 14 libraries (7 replicates).
Results
From the 14 size-selected sequencing libraries, we obtained a total of 101.8 million raw single-end reads, an average of about 7.27 million reads per library. Sequence analysis showed that there was a diverse collection of the exosomal RNA species among which microRNAs (miRNAs) were the most abundant, making up over 42.32% of all raw reads and 76.20% of all mappable reads. At the current read depth, 593 miRNAs were detectable. The five most common miRNAs (miR-99a-5p, miR-128, miR-124-3p, miR-22-3p, and miR-99b-5p) collectively accounted for 48.99% of all mappable miRNA sequences. MiRNA target gene enrichment analysis suggested that the highly abundant miRNAs may play an important role in biological functions such as protein phosphorylation, RNA splicing, chromosomal abnormality, and angiogenesis. From the unknown RNA sequences, we predicted 185 potential miRNA candidates. Furthermore, we detected significant fractions of other RNA species including ribosomal RNA (9.16% of all mappable counts), long non-coding RNA (3.36%), piwi-interacting RNA (1.31%), transfer RNA (1.24%), small nuclear RNA (0.18%), and small nucleolar RNA (0.01%); fragments of coding sequence (1.36%), 5′ untranslated region (0.21%), and 3′ untranslated region (0.54%) were also present. In addition to the RNA composition of the libraries, we found that the three tested commercial kits generated a sufficient number of DNA fragments for sequencing but each had significant bias toward capturing specific RNAs.
Conclusions
This study demonstrated that a wide variety of RNA species are embedded in the circulating vesicles. To our knowledge, this is the first report that applied deep sequencing to discover and characterize profiles of plasma-derived exosomal RNAs. Further characterization of these extracellular RNAs in diverse human populations will provide reference profiles and open new doors for the development of blood-based biomarkers for human diseases.
doi:10.1186/1471-2164-14-319
PMCID: PMC3653748  PMID: 23663360
Exosome; microRNA; Next generation sequencing; Plasma; Biomarker
18.  The Rat Genome Database 2013—data, tools and users 
Briefings in Bioinformatics  2013;14(4):520-526.
The Rat Genome Database (RGD) was started >10 years ago to provide a core genomic resource for rat researchers. Currently, RGD combines genetic, genomic, pathway, phenotype and strain information with a focus on disease. RGD users are provided with access to structured and curated data from the molecular level through the organismal level. Those users access RGD from all over the world. End users are not only rat researchers but also researchers working with mouse and human data. Translational research is supported by RGD’s comparative genetics/genomics data in disease portals, in GBrowse, in VCMap and on gene report pages. The impact of RGD also goes beyond the traditional biomedical researcher, as the influence of RGD reaches bioinformaticians, tool developers and curators. Import of RGD data into other publicly available databases expands the influence of RGD to a larger set of end users than those who avail themselves of the RGD website. The value of RGD continues to grow as more types of data and more tools are added, while reaching more types of end users.
doi:10.1093/bib/bbt007
PMCID: PMC3713714  PMID: 23434633
database; genome; rat; disease; human
19.  Efficient mammalian germline transgenesis by cis-enhanced Sleeping Beauty transposition 
Transgenic research  2010;20(1):29-45.
Heightened interest in relevant models for human disease increases the need for improved methods for germline transgenesis. We describe a significant improvement in the creation of transgenic laboratory mice and rats by chemical modification of Sleeping Beauty transposons. Germline transgenesis in mice and rats was significantly enhanced by in vitro cytosine-phosphodiester-guanine methylation of transposons prior to injection. Heritability of transgene alleles was also greater from founder mice generated with methylated versus non-methylated transposon. The artificial methylation was reprogrammed in the early embryo, leading to founders that express the transgenes. We also noted differences in transgene insertion number and structure (single-insert versus concatemer) based on the influence of methylation and plasmid conformation (linear versus supercoiled), with supercoiled substrate resulting in efficient transpositional transgenesis (TnT) with near elimination of concatemer insertion. Combined, these substrate modifications resulted in increases in both the frequency of transgenic founders and the number of transgenes per founder, significantly elevating the number of potential transgenic lines. Given its simplicity, versatility and high efficiency, TnT with enhanced Sleeping Beauty components represents a compelling non-viral approach to modifying the mammalian germline.
doi:10.1007/s11248-010-9386-5
PMCID: PMC3516389  PMID: 20352328
Sleeping Beauty; Transposon; Transgenesis; Mouse; Rat; Methylation
20.  Creation and Characterization of a Renin Knockout Rat 
Hypertension  2011;57(3):614-619.
The renin-angiotensin system plays an important role in the control of blood pressure (BP) and renal function. To illuminate the importance of renin in the context of a disease background in vivo, we used zinc-finger nucleases (ZFNs) designed to target the renin gene and create a renin knockout in the SS/JrHsdMcwi (SS) rat. ZFN against renin caused a 10-bp deletion in exon 5, resulting in a frameshift mutation. Plasma renin activity was undetectable in the Ren−/− rat, and renin protein was absent from the juxtaglomerular cells in the kidney. Body weight was lower in the Ren−/− rats (than in the Ren+/− or wild-type littermates), and conscious BP on low-salt diet (0.4% NaCl) was 58 ± 2 mm Hg in the Ren−/− male rats versus 117 mm Hg in the Ren+/− littermates, a reduction of almost 50 mm Hg. Blood urea nitrogen (BUN) and plasma creatinine levels were elevated in the Ren−/− strain (BUN 112 ± 7 versus 23 ± 2 mg/dL and creatinine 0.53 ± 0.02 versus 0.26 ± 0.02 mg/dL), and kidney morphology was abnormal with a rudimentary inner renal medulla, cortical interstitial fibrosis, thickening of arterial walls, and abnormally shaped glomeruli. The development of the first rat knockout in the renin-angiotensin system demonstrates the efficacy of the ZFN technology for creating knockout rats for cardiovascular disease on any genetic background and emphasizes the role of renin in BP regulation and kidney function even in the low-renin SS rat.
doi:10.1161/HYPERTENSIONAHA.110.163840
PMCID: PMC3513323  PMID: 21242461
renin; zinc finger nucleases; hypertension; kidney; rat knockout
22.  Endonuclease G is a novel determinant of cardiac hypertrophy and mitochondrial function 
Nature  2011;478(7367):114-118.
Left ventricular mass (LVM) is a highly heritable trait1 and an independent risk factor for all-cause mortality2. To date, genome-wide association studies (GWASs) have not identified the genetic factors underlying LVM variation3 and the regulatory mechanisms for blood pressure (BP)-independent cardiac hypertrophy remain poorly understood4,5. Unbiased systems-genetics approaches in the rat6,7 now provide a powerful complementary tool to GWAS and we applied integrative genomics to dissect a highly replicated, BP-independent LVM locus on rat chromosome 3p. We identified endonuclease G (Endog), previously implicated in apoptosis8 but not hypertrophy, as the gene at the locus and demonstrated loss-of-function mutation in Endog associated with increased LVM and impaired cardiac function. Inhibition of Endog in cultured cardiomyocytes resulted in an increase in cell size and hypertrophic biomarkers in the absence of pro-hypertrophic stimulation. Genome-wide network analysis unexpectedly inferred ENDOG in fundamental mitochondrial processes unrelated to apoptosis. We showed direct regulation of ENDOG by ERRα and PGC1α, master regulators of mitochondrial and cardiac function9,10,11, interaction of ENDOG with the mitochondrial genome and ENDOG-mediated regulation of mitochondrial mass. At baseline, Endog deleted mouse heart had depleted mitochondria, mitochondrial dysfunction and elevated reactive oxygen species (ROS), which was associated with enlarged and steatotic cardiomyocytes. Our studies establish further the link between mitochondrial dysfunction, ROS and heart disease and demonstrate a new role for Endog in maladaptive cardiac hypertrophy.
doi:10.1038/nature10490
PMCID: PMC3189541  PMID: 21979051
23.  Ontology searching and browsing at the Rat Genome Database 
The Rat Genome Database (RGD) is the premier repository of rat genomic and genetic data and currently houses over 40 000 rat gene records, as well as human and mouse orthologs, 1857 rat and 1912 human quantitative trait loci (QTLs) and 2347 rat strains. Biological information curated for these data objects includes disease associations, phenotypes, pathways, molecular functions, biological processes and cellular components. RGD uses more than a dozen different ontologies to standardize annotation information for genes, QTLs and strains. That means a lot of time can be spent searching and browsing ontologies for the appropriate terms needed both for curating and mining the data. RGD has upgraded its ontology term search to make it more versatile and more robust. A term search result is connected to a term browser so the user can fine-tune the search by viewing parent and children terms. Most publicly available term browsers display a hierarchical organization of terms in an expandable tree format. RGD has replaced its old tree browser format with a ‘driller’ type of browser that allows quicker drilling up and down through the term branches, which has been confirmed by testing. The RGD ontology report pages have also been upgraded. Expanded functionality allows more choice in how annotations are displayed and what subsets of annotations are displayed. The new ontology search, browser and report features have been designed to enhance both manual data curation and manual data extraction.
Database URL: http://rgd.mcw.edu/rgdweb/ontology/search.html
doi:10.1093/database/bas016
PMCID: PMC3308169  PMID: 22434847
24.  Gene Targeting in the Rat: Advances and Opportunities 
Trends in genetics : TIG  2010;26(12):510-518.
The rat has long been a model favored by physiologists, pharmacologists, and neuroscientists. However, over the last two decades, many investigators in these fields have turned to the mouse because of its gene modification technologies and extensive genomic resources. While the genomic resources of the rat have nearly caught-up, gene targeting has lagged far behind, limiting the value of the rat for many investigators. In the last two years, advances in transposon- and zinc finger nuclease-mediated gene knockout as well as the establishment and culturing of embryonic and inducible pluripotent stem cells have created new opportunities for rat genetic research. Here, we provide a high-level description and potential uses of these new technologies for investigators using the rat for biomedical research.
doi:10.1016/j.tig.2010.08.006
PMCID: PMC2991520  PMID: 20869786
25.  RGD: A comparative genomics platform 
Human genomics  2011;5(2):124-129.
The Rat Genome Database (RGD) (http://rgd.mcw.edu) provides a comprehensive platform for comparative genomics and genetics research. RGD houses gene, QTL and polymorphic marker data for rat, mouse and human and provides easy access to data through sophisticated searches, disease portals, interactive pathway diagrams and rat and human genome browsers.
PMCID: PMC3222152  PMID: 21296746
genomics; database; disease

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