Ratpost: a searchable database of protein-inactivating sequence variations in 40 sequenced rat-inbred strains.

Rat-inbred strains are essential as scientific tools. We have analyzed the publicly available genome sequences of 40 rat-inbred strains and provide an overview of sequence variations leading to amino acid changes in protein-coding genes, premature STOP codons or loss of STOP codons, and short in-frame insertions and deletions of all protein-coding genes across all these inbred lines. We provide an overview of the predicted impact on protein function of all these affected proteins in the database, by comparing their sequence with the sequences of the rat reference strain BN/SsNHsdMcwi. We also investigate the flaws of the protein-coding sequences of this reference strain itself, by comparing them with a consensus genome. These data can be retrieved via a searchable website (Ratpost.be) and allow a global, better interpretation of genetic background effects and a source of naturally defective alleles in these 40 sequenced classical and high-priority rat-inbred strains.

Voluntary exposure to a toxin: the genetic influence on ethanol consumption.

Ethyl alcohol is a toxin that, when consumed at high levels, produces organ damage and death. One way to prevent or ameliorate this damage in humans is to reduce the exposure of organs to alcohol by reducing alcohol ingestion. Both the propensity to consume large volumes of alcohol and the susceptibility of human organs to alcohol-induced damage exhibit a strong genetic influence. We have developed an integrative genetic/genomic approach to identify transcriptional networks that predispose complex traits, including propensity for alcohol consumption and propensity for alcohol-induced organ damage. In our approach, the phenotype is assessed in a panel of recombinant inbred (RI) rat strains, and quantitative trait locus (QTL) analysis is performed. Transcriptome data from tissues/organs of naïve RI rat strains are used to identify transcriptional networks using Weighted Gene Coexpression Network Analysis (WGCNA). Correlation of the first principal component of transcriptional coexpression modules with the phenotype across the rat strains, and overlap of QTLs for the phenotype and the QTLs for the coexpression modules (module eigengene QTL) provide the criteria for identification of the functionally related groups of genes that contribute to the phenotype (candidate modules). While we previously identified a brain transcriptional module whose QTL overlapped with a QTL for levels of alcohol consumption in HXB/BXH RI rat strains and 12 selected rat lines, this module did not account for all of the genetic variation in alcohol consumption. Our search for QTL overlap and correlation of coexpression modules with phenotype can, however, be applied to any organ in which the transcriptome has been measured, and this represents a holistic approach in the search for genetic contributors to complex traits. Previous work has implicated liver/brain interactions, particularly involving inflammatory/immune processes, as influencing alcohol consumption levels. We have now analyzed the liver transcriptome of the HXB/BXH RI rat panel in relation to the behavioral trait of alcohol consumption. We used RNA-Seq and microarray data to construct liver transcriptional networks, and identified a liver candidate transcriptional coexpression module that explained 24% of the genetic variance in voluntary alcohol consumption. The transcripts in this module focus attention on liver secretory products that influence inflammatory and immune signaling pathways. We propose that these liver secretory products can interact with brain mechanisms that affect alcohol consumption, and targeting these pathways provides a potential approach to reducing high levels of alcohol intake and also protecting the integrity of the liver and other organs.

Development and Characterization of a Novel Congenic Rat Strain for Obesity and Cancer Research.

The association between a Western Diet and colon cancer suggests that dietary factors and/or obesity may contribute to cancer progression. Our objective was to develop a new animal model of obesity and the associated pathophysiology to investigate human cancer independent of dietary components that induce obesity. A novel congenic rat strain was established by introducing the fa allele from the Zucker rat into the Rowett Nude rat to generate a "fatty nude rat". The obese phenotype was first characterized in the new model. To then examine the utility of this model, lean and obese rats were implanted with HT-29 human colon cancer xenografts and tumor growth monitored. Fatty nude rats were visibly obese and did not develop fasting hyperglycemia. Compared to lean rats, fatty nude rats developed fasting hyperinsulinemia, glucose intolerance, and insulin resistance. Colon cancer tumor growth rate and final weight were increased (P < 0.05) in fatty nude compared to lean rats. Final tumor weight was associated with p38 kinase phosphorylation (P < 0.01) in fatty nude rats. We have established a novel model of obesity and pre-type 2 diabetes that can be used to investigate human cancer and therapeutics in the context of obesity and its associated pathophysiology.

[THE ISRAELI RAT GENOME CENTER].

INTRODUCTION: The Israeli Rat Genome Center, which is located at the Barzilai Medical Center Campus of the Faculty of Health Sciences of the Ben-Gurion University of the Negev in Ashkelon, was established to provide a repository of unique genetic strains of rats that were created in Israel and that simulate complex diseases. The Center incorporates models of: salt-sensitive hypertension (SBN/y and SBH/y rats), type 2 diabetes (CDr and CDs rats), combined hypertension and diabetes (CRDH) and additional genetic strains (transgenics, consomics, congenics). All these strains are available to researchers who are interested in the study of complex diseases, on the basis of collaboration. The Laboratory for Molecular Medicine within the Center also performs independent research on the genetic basis of complex diseases, including hypertension, diabetes, kidney disease and target organ involvement in cardiovascular and metabolic diseases. The Center collaborates with researchers from research centers in other medical centers and universities in Israel and worldwide.

Rat models of cardiometabolic diseases: baseline clinical chemistries, and rationale for their use in examining air pollution health effects.

Individuals with cardiovascular and metabolic diseases (CVD) are shown to be more susceptible to adverse health effects of pollutants. Rodent models of CVD are used for examining susceptibility variations. CVD models developed by selective inbreeding are shown to represent the etiology of human disease and metabolic dysfunction. The goal of this article was to review the origin and the pathobiological features of rat models of varying CVD with or without metabolic syndrome and healthy laboratory rat strains to allow better interpretation of the data regarding their susceptibility to air pollutant exposures. Age-matched healthy Sprague-Dawley (SD), Wistar (WIS) and Wistar Kyoto (WKY), and CVD-prone spontaneously hypertensive (SH), Fawn-Hooded hypertensive (FHH), SH stroke-prone (SHSP), SHHF/Mcc heart failure obese (SHHF) and insulin-resistant JCR:LA-cp obese (JCR) rat models were considered for this study. The genetics and the underlying pathologies differ between these models. Normalized heart weights correlated with underlying cardiac disease while wide differences exist in the number of white blood cells and platelets within healthy strains and those with CVD. High plasma fibrinogen and low angiotensin converting enzyme activity in FHH might relate to kidney disease and associated hypertension. However, other obese strains with known kidney lesions do not exhibit decreases in ACE activity. The increased activated partial thromboplastin time only in SHSP correlates with their hemorrhagic stroke susceptibility. Increases plasma lipid peroxidation in JCR might reflect their susceptibility to acquire atherosclerosis. These underlying pathologies involving CVD and metabolic dysfunction are critical in interpretation of findings related to susceptibility variations of air pollution health effects.

Genetic Relatedness of WNIN and WNIN/Ob with Major Rat Strains in Biomedical Research.

WNIN (Wistar/NIN) is an inbred rat strain maintained at National Institute of Nutrition (NIN) for more than 90 years, and WNIN/Ob is an obese mutant originated from it. To determine their genetic relatedness with major rat strains in biomedical research, they were genotyped at various marker loci. The recently identified markers for albino and hooded mutations which clustered all the known albino rats into a single lineage also included WNIN and WNIN/Ob rats. Genotyping using microsatellite DNA markers and phylogenetic analysis with 49 different rat strains suggested that WNIN shares a common ancestor with many Wistar originated strains. Fst estimates and Fischer's exact test suggest that WNIN rats differed significantly from all other strains tested. WNIN/Ob though shows hyper-leptinemia, like Zucker fatty rat, did not share the Zucker fatty rat mutation. The above analyses suggest WNIN as a highly differentiated rat strain and WNIN/Ob a novel obese mutant evolved from it.

Development and characterization of a new inbred transgenic rat strain expressing DsRed monomeric fluorescent protein.

The inbred rat is a suitable model for studying human disease and because of its larger size is more amenable to complex surgical manipulation than the mouse. While the rodent fulfills many of the criteria for transplantation research, an important requirement is the ability to mark and track donors cells and assess organ viability. However, tracking ability is limited by the availability of transgenic (Tg) rats that express suitable luminescent or fluorescent proteins. Red fluorescent protein cloned from Discosoma coral (DsRed) has several advantages over other fluorescent proteins, including in vivo detection in the whole animal and ex vivo visualization in organs as there is no interference with autofluorescence. We generated and characterized a novel inbred Tg Lewis rat strain expressing DsRed monomeric (DsRed mono) fluorescent protein under the control of a ubiquitously expressed ROSA26 promoter. DsRed mono Tg rats ubiquitously expressed the marker gene as detected by RT-PCR but the protein was expressed at varying levels in different organs. Conventional skin grafting experiments showed acceptance of DsRed monomeric Tg rat skin on wild-type rats for more than 30 days. Cardiac transplantation of DsRed monomeric Tg rat hearts into wild-type recipients further showed graft acceptance and long-term organ viability (>6 months). The DsRed monomeric Tg rat provides marked cells and/or organs that can be followed for long periods without immune rejection and therefore is a suitable model to investigate cell tracking and organ transplantation.

A novel active endogenous retrovirus family contributes to genome variability in rat inbred strains.

Endogenous retroviruses (ERVs) contribute to a range of germline, as well as somatic mutations in mammals. However, autonomous retrotransposition of potentially active elements has not been demonstrated in the rat genome. We cloned an insertion that disrupted the normal splicing of the Cntrob gene that was subsequently identified as a nonautonomous, novel endogenous retrovirus of the RnERV-K8e family. The RnERV-K8e family is closely related to the recently reported MmERV-K10c elements, but differs from the autonomous mouse MusD or IAP families. In addition, we identified a novel, unexpectedly close relative of RnERV-K8e in the mouse, suggesting ERV-K cross-species transmission between mice and rats. We cloned a potentially autonomous RnERV-K8e element identified by in silico analysis and, using an in vitro retrotransposition assay, demonstrated that it is capable of retrotransposition. This particular element (named Rat-rho, pronounced "retro") encodes a retroviral envelope gene (env); however, env is not required for de novo retrotransposition events. Significant levels of RnERV-K8e-associated genetic polymorphisms were detected among inbred rat strains, suggesting ongoing retrotransposition in the rat genome. This study identifies an ERV-K-type family in rats that shows obvious signs of recent activity. Ongoing retrotranspositional activity may significantly add to genomic variability among inbred rat strains.

Genetic and phenotypic characterization of a Japanese wild-derived DOB/Oda rat strain.

Wild-derived rat strains can provide novel genome resources that are not available in standard laboratory strains. Genetic backgrounds of wild-derived strains can facilitate effective genetic linkage analyses and often modulate the expression of mutant phenotypes. Here we describe the development and characterization of a new inbred rat strain, DOB/Oda, from wild rats (Rattus norvegicus) captured in Shitara, Aichi, Japan. Phenotype analysis of 109 parameters revealed that the DOB/Oda rats had small body weight, preference for darkness, and high locomotor activity compared with the rat strains in the National BioResource Project for the Rat (NBRP-Rat) database. Genome analysis with 357 SSLP markers identified DOB/Oda-specific alleles in 70 markers. The percentage of SSLP markers that showed polymorphism between the DOB/Oda strain and any of 132 laboratory strains from NBRP-Rat varied from 89 to 95 %. The polymorphic rate (average of the values of the percentage) for the DOB/Oda strain was 91.6 %, much higher than the rates for available wild-derived strains such as the Brown Norway rat. A phylogenic tree constructed with DOB/Oda and all the strains in NBRP-Rat showed that the DOB/Oda strain localized within the wild rat groups, apparently separate from the laboratory strains. Together, these findings indicated that the DOB/Oda rat has a unique genome that is not available in the laboratory strains. Therefore, the new DOB/Oda strain will provide an important genome resource that will be useful for designing genetic experiments and for the discovery of genes that modulate mutant phenotypes.

The case for genetic monitoring of mice and rats used in biomedical research.

Currently, there is the potential to generate over 200,000 mutant mouse strains between existing mouse strains (over 24,000) and genetically modified mouse embryonic stem cells (over 209,000) that have been entered into the International Mouse Strain Resource Center (IMSR) from laboratories and repositories all over the world. The number of rat strains is also increasing exponentially. These mouse and rat mutants are a tremendous genetic resource; however, the awareness of their genetic integrity such as genetic background and genotyping of these models is not always carefully monitored. In this review, we make a case for the International Council for Laboratory Animal Science (ICLAS), which is interested in promoting and helping academic institutions develop a genetic monitoring program to bring a level of genetic quality assurance into the scientific interchange and use of mouse and rat genetically mutant models.

Strain influences on inflammatory pathway activation, cell infiltration and complement cascade after traumatic brain injury in the rat.

Increasing evidence suggests that genetic background affects outcome of traumatic brain injuries (TBI). Still, there is limited detailed knowledge on what pathways/processes are affected by genetic heterogeneity. The inbred rat strains DA and PVG differ in neuronal survival following TBI. We here carried out global expressional profiling to identify differentially regulated pathways governing the response to an experimental controlled brain contusion injury. One of the most differentially regulated molecular networks concerned immune cell trafficking. Subsequent characterization of the involved cells using flow cytometry demonstrated greater infiltration of neutrophils and monocytes, as well as a higher degree of microglia activation in DA compared to PVG rats. In addition, DA rats displayed a higher number of NK cells and a higher ratio of CD161bright compared to CD161dim NK cells. Local expression of complement pathway molecules such as C1 and C3 was higher in DA and both the key complement component C3 and membrane-attack complex (MAC) could be demonstrated on axons and nerve cells. A stronger activation of the complement system in DA was associated with higher cerebrospinal fluid levels of neurofilament-light, a biomarker for nerve/axonal injury. In summary, we demonstrate substantial differences between DA and PVG rats in activation of inflammatory pathways; in particular, immune cell influx and complement activation associated with neuronal/axonal injury after TBI. These findings suggest genetic influences acting on inflammatory activation to be of importance in TBI and motivate further efforts using experimental forward genetics to identify genes/pathways that affect outcome.

Cosegregation of blood pressure with angiotensin converting enzyme and atrial natriuretic peptide receptor genes using Dahl salt-sensitive rats.

We have evaluated the genes for angiotensin converting enzyme (ACE) and guanylyl cyclase A/atrial natriuretic peptide receptor (GCA) for genetic effects on blood pressure response to high salt diet. In F2 rats derived from Milan normotensive and Dahl salt-hypertension sensitive (S) rats, both ACE and GCA cosegregated with blood pressure, and rats that were homozygous for the S allele at both the ACE and GCA loci had inordinately high blood pressure. In F2 derived from Wistar Kyoto (WKY) and S rats, GCA revealed positive cosegregation with blood pressure, but ACE did not. We conclude that certain alleles at the GCA and ACE loci (or at loci closely linked to them) have a significant genetic impact on blood pressure response to high salt in specific rat strains.

Mutations in MERTK, the human orthologue of the RCS rat retinal dystrophy gene, cause retinitis pigmentosa.

Mutation of a receptor tyrosine kinase gene, Mertk, in the Royal College of Surgeons (RCS) rat results in defective phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) and retinal degeneration. We screened the human orthologue, MERTK, located at 2q14.1 (ref. 10), in 328 DNA samples from individuals with various retinal dystrophies and found three mutations in three individuals with retinitis pigmentosa (RP). Our findings are the first conclusive evidence implicating the RPE phagocytosis pathway in human retinal disease.

Genetic influences on survival time after severe hemorrhage in inbred rat strains.

To find a genetic basis for differential ability to survive severe hemorrhage, we previously showed eightfold differences in survival times among inbred rat strains. We assumed that rat strains had similar normalized blood volumes (NBV; ml/100 g body wt). As NBV might vary among strains and constitute one genetic variable affecting survival time to hemorrhage, in experiment 1 of the current studies we first measured total blood volumes and calculated NBV in specific inbred rat strains (Brown Norway/Medical College of Wisconsin, BN; Dark Agouti, DA; Fawn Hooded Hypertensive, FHH; Lewis, LEW; and Dahl Salt-Sensitive, SS) previously found to be divergent in survival time. NBV differed by 20% (P < 0.01; BN > SS > FHH = LEW = DA) and had a heritability (h(2)) of 0.56. Hence, differential survival times in our previously published study might reflect strain-dependent differences in NBV. Then studies were conducted wherein rats were catheterized and, ∼24 h later, 47% of their blood volume was removed; these rats were observed for a maximum of 4 h. In experiment 2, blood volumes were measured the day prior to hemorrhage. Percent survival and survival time did not differ among strains. To obviate possible confounding effects of blood volume determination, in experiment 3 the average NBV for each strain was used to determine hemorrhage volumes. Percent survival (P < 0.01) and survival times (P < 0.001) were different with DA demonstrating the best (62.5%, 190 ± 29 min) and BN the worst (0%, 52 ± 5 min) survival responses. These data indicate that both blood volume and survival time after hemorrhage in rats are heritable quantitative traits, and continue to suggest that genetic assessment of these phenotypes might lead to novel therapeutics to improve survival to hemorrhage.

Genetic resistance to the induction of experimental allergic encephalomyelitis in Lewis rats. I. Genetic analysis of an apparent mutant strain with phenotypic resistance to experimental allergic encephalomyelitis.

Clinical resistance to the induction of experimental allergic encephalomyelitis was observed in a closed colony of Lewis (designated Le-R) rats. Disease susceptibility in randomly bred animals appeared to increase with increasing age. In the small group of young Le-R rats, which were susceptible, disease onset was delayed, severity of symptoms was reduced, and duration of clinical signs was abbreviated compared to conventional Lewis rats. The severity of histologic neural tissue lesions correlated with clinical observations. Breeding experiments indicated that most Le-R rats were resistant to disease induction regardless of whether their ancestors had been selected for susceptibility or resistance. The F3 generation of resistant lineage was uniformly resistant at all ages tested. Virtually all (Lewis X Le-R)F1 rats of either sex were resistant when challenged at 7-8 wk of age indicating that resistance was a dominant autosomal trait. Approximately half of (F1 X Lewis) backcross rats developed paralytic EAE whereas one-fourth were entirely resistant, suggesting that disease resistance may be mediated by one or two genes. Le-R rats shared at least some of the Lewis rat major histocompatibility antigens. Resistance apparently did not reflect a nonspecific impairment of cellular immune responsiveness. Le-R rats, which had been challenged with myelin basic protein, developed antigen-reactive cells specific for basic protein or its encephalitogenic fragment. Spleen cells obtained from basic protein-sensitized Le-R rats did not adoptively transfer disease into Lewis rats. In contrast, spleen cells obtained from basic protein-sensitized Lewis rats readily transferred disease into both Lewis and Le-R recipients. These data suggest that disease resistance may be a result of an immunologic deficit (or suppressor cell activity) expressed during the differentiation of antigen-reactive cells into disease-inducing effector cells.

Recent advances in genetics of the spontaneously hypertensive rat.

The spontaneously hypertensive rat (SHR) is the most widely used animal model of essential hypertension and associated metabolic disturbances. Multiple quantitative trait loci associated with hemodynamic and metabolic parameters have been mapped in the SHR. Recently, it has become possible to identify some of the specific quantitative trait gene (QTG) variants that underlie quantitative trait loci linked to complex cardiovascular and metabolic traits in SHR related strains. Recombinant inbred strains derived from SHR and Brown Norway progenitors, together with SHR congenic and transgenic strains, have proven useful for establishing the identity of several QTGs in SHR models. It is anticipated that the combined use of linkage analyses and gene expression profiles, together with the recently available genome sequences of both the SHR and Brown Norway strains and new methods for manipulating the rat genome, will soon accelerate progress in identifying QTGs for complex traits in SHR-related strains.

Calcium channel dysfunction in inferior colliculus neurons of the genetically epilepsy-prone rat.

Voltage-gated calcium (Ca(2+)) channels are thought to play an important role in epileptogenesis and seizure generation. Here, using the whole cell configuration of patch-clamp techniques, we report on the modifications of biophysical and pharmacological properties of high threshold voltage-activated Ca(2+) channel currents in inferior colliculus (IC) neurons of the genetically epilepsy-prone rats (GEPR-3s). Ca(2+) channel currents were measured by depolarizing pulses from a holding potential of - 80 mV using barium (Ba(2+)) as the charge carrier. We found that the current density of high threshold voltage-activated Ca(2+) channels was significantly larger in IC neurons of seizure-naive GEPR-3s compared to control Sprague-Dawley rats, and that seizure episodes further enhanced the current density in the GEPR-3s. The increased current density was reflected by both a - 20 mV shifts in channel activation and a 25% increase in the non-inactivating fraction of channels in seizure-naive GEPR-3s. Such changes were reduced by seizure episodes in the GEPR-3s. Pharmacological analysis of the current density suggests that upregulation of L-, N- and R-type of Ca(2+) channels may contribute to IC neuronal hyperexcitability that leads to seizure susceptibility in the GEPR-3s.

[Individual and strain differences of aldehyde oxidase in the rat].

Together with xanthine oxidase, aldehyde oxidase (AO) is a major member of a relatively small family of molybdenum hydroxylases. Both enzymes are homodimers with a subunit molecular weight of about 150 kDa and exhibit catalytic activity only as a dimer. An AO subunit contains a molybdopterin cofactor, an FAD and two different 2Fe-2S redox centers. The enzyme catalyzes oxidation of a wide range of endogenous and exogenous aldehydes and N-heterocyclic aromatic compounds. N-heterocycle-containing drugs such as methotrexate, 6-mercaptopurine, cinchona alkaloids and famciclovir are oxidized by this enzyme. Marked species differences have been well documented for the AO-catalyzed metabolism of drugs including methotrexate and famciclovir. In addition, a large rat strain variation has also been demonstrated in the oxidation activity of benzaldehyde and methotrexate. Marked differences in species, large differences in rat strains and individual differences in AO activities in some rat strains have been reported. However, little has been elucidated about any related molecular biological mechanisms. We examined the mechanism of individual variations and strain difference of rat AO using the technology of molecular biology. Our recent studies regarding the inter- and intra-difference of AO activities in rats are described.

The Roman High- and Low-Avoidance rat strains differ in fear-potentiated startle and classical aversive conditioning.

The Swiss sublines of Roman High-(RHA/Verh) and Low-(RLA/Verh) Avoidance rats have been genetically selected (and outbred) since 1972 because of their good versus extremely poor acquisition of two-way, active avoidance. Inbred strains (RHA-I and RLA-I), derived from those two lines, have been maintained at our laboratory since 1997. The RLA line/strain shows increased stress-induced endocrine responses and enhanced anxiety/fearfulness in a variety of unconditioned behavioural variables and tests. Thus far, however, the Roman rat strains have not been compared in procedures involving classical fear conditioning to cues or contexts. Therefore, the present work was aimed at comparing RHA-I and RLA-I rats in 1) two different procedures of fear-potentiated startle and 2) in a classical fear conditioning (i.e., conditioned freezing) paradigm. The results indicate that, compared to RHA-I rats, RLA-I animals display higher levels of conditioned fear (as measured either by startle responses or freezing behavior) across those different tasks.

Extended regions of suspected mis-assembly in the rat reference genome.

We performed whole-genome sequencing for eight inbred rat strains commonly used in genetic mapping studies. They are the founders of the NIH heterogeneous stock (HS) outbred colony. We provide their sequences and variant calls to the rat genomics community. When analyzing the variant calls we identified regions with unusually high levels of heterozygosity. These regions are consistent across the eight inbred strains, including Brown Norway, which is the basis of the rat reference genome. These regions show higher read depths than other regions in the genome and contain higher rates of apparent tri-allelic variant sites. The evidence suggests that these regions may correspond to duplicated segments that were incorrectly overlaid as a single segment in the reference genome. We provide masks for these regions of suspected mis-assembly as a resource for the community to flag potentially false interpretations of mapping or functional results.