Prior to the advent of genetic engineering in the mouse, the rat was the model of choice for investigating the etiology of cancer. Now, recent advances in the manipulation of the rat genome, combined with a growing recognition of the physiological differences between mice and rats, have reignited interest in the rat as a model of human cancer. Two recently developed rat models, the polyposis in the rat colon (Pirc) and Kyoto Apc Delta (KAD) strains, each carry mutations in the intestinal-cancer-associated adenomatous polyposis coli (Apc) gene. In contrast to mouse models carrying Apc mutations, in which cancers develop mainly in the small intestine rather than in the colon and there is no gender bias, these rat models exhibit colonic predisposition and gender-specific susceptibility, as seen in human colon cancer. The rat also provides other experimental resources as a model organism that are not provided by the mouse: the structure of its chromosomes facilitates the analysis of genomic events, the size of its colon permits longitudinal analysis of tumor growth, and the size of biological samples from the animal facilitates multiplexed molecular analyses of the tumor and its host. Thus, the underlying biology and experimental resources of these rat models provide important avenues for investigation. We anticipate that advances in disease modeling in the rat will synergize with resources that are being developed in the mouse to provide a deeper understanding of human colon cancer.

The early loss of photoreceptors in some retinal degenerations in mice has been shown to have a profound effect on vascular development of the retina. To better characterize this relationship, we have examined the formation of retinal blood vessels during the first month of life in 8 lines of transgenic rats with different ages of onset and rates of photoreceptor cell loss mediated by the expression of mutant rhodopsin (P23H and S334ter). The number of capillary profiles in the superficial plexus (SP) and deep capillary plexus (DCP) of the retina were quantified in retinal sections taken at postnatal day (P) 8, 10, 12, 15 and 30. In normal wild-type rats, the SP and DCP had mostly established mature, adult patterns by P15, as previously shown. In the transgenic rats, the loss of photoreceptors had relatively little effect on the SP. By contrast, the loss of photoreceptors during vascular development had a major impact on the DCP. In the two lines with early and most rapid photoreceptor loss, S334ter-7 and S334ter-3, where about 90% and 65%, respectively, of the photoreceptors were already lost by P15, the DCP either failed to form (S334ter-7) or the number of capillary profiles was less than 7% of controls (S334ter-3). In lines where almost all photoreceptors were still present at P15 (S334ter-4, S334ter-9, P23H-2 and P23H-3), the number of profiles in the DCP were the same as in wild-type controls at P30. In two lines with an intermediate rate of degeneration (S334ter-5 and P23H-1), where only about 25% of the photoreceptors were lost by P15, there was an intermediate number of vascular profiles in the DCP at P30. Thus, a very close relationship between the number of photoreceptors and vessel profiles in the DCP during its development exists in the transgenic rats, and the loss of photoreceptors results in the failure or inhibition of the DCP to develop. Several mechanisms may explain this relationship including changes in the level of physiological oxygen tension or alteration in the release of angiogenic factors that normally drive vessel development. Analysis of older transgenic retinas up to 1 year of age revealed that (1) vascular profiles are lost from the DCP in essentially all lines once fewer than about 30-33% of photoreceptors remain; (2) in those lines where the DCP essentially did not develop (S334ter-7 and S334ter-3), the effect of photoreceptor absence was permanent, and there was no late vascularization of the DCP; (3) the number of capillary profiles in the SP remained no different from controls in any of the lines, despite long-standing loss of photoreceptors; and (4) neovascularization of the RPE by retinal capillaries occurred with a latency of 60-180 days after the loss of photoreceptors, except in S334ter-7 rats, where neovascularization essentially did not occur. Analysis of RCS rats was carried out for comparison.

The Matsumoto Eosinophilia Shinshu (MES) is a rat model for hereditary blood eosinophilia. The incidence of eosinophilia is 100% in both female and male MES. The primary cause of the eosinophilia in MES is a loss-of-function mutation in the gene encoding the cytochrome b-245, alpha polypeptide (Cybames mutant allele). CYBA protein is a constituent of the superoxide-generating NADPH oxidase complex, the catalytic subunit of which is either NOX1, NOX2, or NOX4. However, the molecular mechanisms for the loss of CYBA to cause eosinophilia and even which of the three NOX isotypes is causally linked to the disease have been unknown. To resolve the latter issue, we generated F344/N rats knockout for Nox1, Nox2, and Nox4 genes. Also, we bred F344.MES-Cybames congenic rats that have a similar genetic background to the Nox knockout rats. We found that approximately 20% of female F344/N-Nox2em1 rats but none of the males developed blood eosinophilia. Also, we observed that all female F344.MES-Cybames and approximately 50% of male congenic rats developed the disorder. These results revealed that loss of NOX2 is the cause of blood eosinophilia in rats. Meanwhile, the data also indicated that in addition to the loss of NOX2 NADPH oxidase, both the genetic background of F344/N strain and gender influence the development of the disorder. These Nox and Cyba mutant rat strains with different eosinophilia incidences should be useful to elucidate molecular mechanisms and factors involved in the development of the disease.

It has been shown that B cell activating factor receptor (BAFFR) is critical for B cell development and survival. In this study, we sought to evaluate the expression and function of BAFFR across multiple stains of mice that vary in their potential to develop systemic autoimmune disease. The inability of a commercial antibody to bind to BAFFR in the autoimmune prone mouse strains, MRL and MRL/Lpr led to the discovery of a mutation in TNFRSF13C gene (encoding BAFFR) that resulted in a Pro44Ser substitution in the N-terminus near the BAFF binding site in these strains. To define the biological consequences of mutant BAFFR, we compared the expression and activity of BAFFR in MRL and MRL/Lpr mice to BALB/c, which express the consensus version of TNFRSF13C. B cells from MRL and MRL/Lpr mice expressed mutant BAFFR on surface and were capable of responding to BAFF as exhibited by BAFF-mediated reduction in apoptosis and NF-κB2 activation. Signaling through MAPK ERK1/2 was not significantly induced by BAFF in MRL/Lpr mice; however, MAPK ERK1/2 signaling was intact in MRL mice. The inability of MRL/Lpr B cells to significantly activate ERK1/2 in response to BAFF was due to the high basal activity of the signaling pathway in these cells. In fact, basal activity of ERK1/2 in B cells correlated with the degree of autoimmune susceptibility exhibited by each strain. In addition, aged MRL/Lpr mice with severe autoimmune disease had high BAFF levels, low surface BAFFR, and high basal NF-κB2 activation, a pattern which is attributed to the high frequency of antibody secreting cells. We conclude that P44S BAFFR mutation does not hinder BAFFR function or enhance B cell activity in MRL/Lpr and MRL mice and that other susceptibility loci on the MRL background contributed to the hyperactivity of these cells.

Mutations in p53, although frequent in human cancers, have not been implicated in telomere-related syndromes. Here, we show that homozygous mutant mice expressing p53Δ31, a p53 lacking the C-terminal domain, exhibit increased p53 activity and suffer from aplastic anemia and pulmonary fibrosis, hallmarks of syndromes caused by short telomeres. Indeed, p53Δ31/Δ31 mice had short telomeres and other phenotypic traits associated with the telomere disease dyskeratosis congenita and its severe variant the Hoyeraal-Hreidarsson syndrome. Heterozygous p53+/Δ31 mice were only mildly affected, but decreased levels of Mdm4, a negative regulator of p53, led to a dramatic aggravation of their symptoms. Importantly, several genes involved in telomere metabolism were downregulated in p53Δ31/Δ31 cells, including Dyskerin, Rtel1, and Tinf2, which are mutated in dyskeratosis congenita, and Terf1, which is implicated in aplastic anemia. Together, these data reveal that a truncating mutation can activate p53 and that p53 plays a major role in the regulation of telomere metabolism.

We illustrate the growing power of the BXD family of mice (recombinant inbred strains from a cross of C57BL/6J and DBA/2J mice) and companion bioinformatic tools to study complex genome-phenome relations related to glaucoma. Over the past 16 years, our group has integrated powerful murine resources and web-accessible tools to identify networks modulating visual system traits-from photoreceptors to the visual cortex. Recent studies focused on retinal ganglion cells and glaucoma risk factors, including intraocular pressure (IOP), central corneal thickness (CCT), and susceptibility of cellular stress. The BXD family was exploited to define key gene variants and then establish linkage to glaucoma in human cohorts. The power of this experimental approach to precision medicine is highlighted by recent studies that defined cadherin 11 (Cdh11) and a calcium channel (Cacna2d1) as genes modulating IOP, Pou6f2 as a genetic link between CCT and retinal ganglion cell (RGC) death, and Aldh7a1 as a gene that modulates the susceptibility of RGCs to death after elevated IOP. The role of three of these gene variants in glaucoma is discussed, along with the pathways activated in the disease process.

The laboratory rat (Rattus norvegicus) is a key model organism for biomedical research. Rats can be subjected to strict genetic and environmental controls. The rat's large body size is suitable for both surgical operations and repeated measurements of physiological parameters. These advantages have led to the development of numerous rat models for genetic diseases. Forward genetics is a proven approach for identifying the causative genes of these disease models but requires genome resources including genetic markers and genome sequences. Over the last few decades, rat genome resources have been developed and deposited in bioresource centers, which have enabled us to perform positional cloning in rats. To date, more than 100 disease-related genes have been identified by positional cloning. Since some disease models are more accessible in rats than mice, the identification of causative genes in these models has sometimes led to the discovery of novel functions of genes. As before, various mutant rats are also expected to be discovered and developed as disease models in the future. Thus, the forward genetics continues to be an important approach to find genes involved in disease phenotypes in rats. In this review, I provide an overview the development of rat genome resources and describe examples of positional cloning in rats in which novel gene functions have been identified.

Protein misfolding caused by inherited mutations leads to loss of protein function and potentially toxic 'gain of function', such as the dominant P23H rhodopsin mutation that causes retinitis pigmentosa (RP). Here, we tested whether the AMPK activator metformin could affect the P23H rhodopsin synthesis and folding. In cell models, metformin treatment improved P23H rhodopsin folding and traffic. In animal models of P23H RP, metformin treatment successfully enhanced P23H traffic to the rod outer segment, but this led to reduced photoreceptor function and increased photoreceptor cell death. The metformin-rescued P23H rhodopsin was still intrinsically unstable and led to increased structural instability of the rod outer segments. These data suggest that improving the traffic of misfolding rhodopsin mutants is unlikely to be a practical therapy, because of their intrinsic instability and long half-life in the outer segment, but also highlights the potential of altering translation through AMPK to improve protein function in other protein misfolding diseases.

Modern molecular genetics create a rapidly growing number of mutant mouse lines, many of which need to be phenotyped behaviorally. Poor reliability and low efficiency of traditional behavioral tests have prompted the development of new approaches to behavioral phenotyping, such as fully automated analysis of behavior in the homecage.

The shaker rat is an X-linked recessive spontaneous model of progressive Purkinje cell (PC) degeneration exhibiting a shaking ataxia and wide stance. Generation of Wistar Furth (WF)/Brown Norwegian (BN) F1 hybrids and genetic mapping of F2 sib-sib offspring using polymorphic markers narrowed the candidate gene region to 26 Mbp denoted by the last recombinant genetic marker DXRat21 at 133 Mbp to qter (the end of the long arm). In the WF background, the shaker mutation has complete penetrance, results in a stereotypic phenotype and there is a narrow window for age of disease onset; by contrast, the F2 hybrid phenotype was more varied, with a later age of onset and likely non-penetrance of the mutation. By deep RNA-sequencing, five variants were found in the candidate region; four were novel without known annotation. One of the variants caused an arginine (R) to cysteine (C) change at codon 35 of the ATPase, Ca(2+) transporting, plasma membrane 3 (Atp2b3) gene encoding PMCA3 that has high expression in the cerebellum. The variant was well supported by hundreds of overlapping reads, and was found in 100% of all affected replicas and 0% of the wild-type (WT) replicas. The mutation segregated with disease in all affected animals and the amino acid change was found in an evolutionarily conserved region of PMCA3. Despite strong genetic evidence for pathogenicity, in vitro analyses of PMCA3(R35C) function did not show any differences to WT PMCA3. Because Atp2b3 mutation leads to congenital ataxia in humans, the identified Atp2b3 missense change in the shaker rat presents a good candidate for the shaker rat phenotype based on genetic criteria, but cannot yet be considered a definite pathogenic variant owing to lack of functional changes.

No abstract available

Neurological and psychiatric disorders are among the most common and most serious health problems in developed countries. Transgenic mouse models mimicking human neurological diseases have provided new insights into development and function of the nervous system. One of the prominent goals of the German National Genome Research Network is the understanding of the in vivo function of single genes and the pathophysiological and clinical consequences of respective mutations. The German Mouse Clinic (GMC) offers a high-throughput primary screen of genetically modified mouse models as well as an in-depth analysis in secondary and tertiary screens covering various fields of mouse physiology. Here we describe the phenotyping methods of the Neurological Screen in the GMC, exemplified in the four inbred mouse lines C57BL/6J, C3HeB/FeJ, BALB/cByJ, and 129S2/SvPas. For our primary screen, we generated "standard operating procedures" that were validated between different laboratories. The phenotyping of inbred strains already showed significant differences in various parameters, thus being a prerequisite for the examination of mutant mouse lines.

The mouse has emerged as a uniquely valuable species for studying the molecular and genetic basis of complex behaviors and modeling neuropsychiatric disease states. While valid and reliable preclinical assays for reward-related behaviors are critical to understanding addiction-related processes, and various behavioral procedures have been developed and characterized in rats and primates, there have been relatively few studies using operant-based addiction-relevant behavioral paradigms in the mouse. Here we describe the performance of the C57BL/6J inbred mouse strain on three major reward-related paradigms, and replicate the same procedures in two other commonly used inbred strains (DBA/2J, BALB/cJ). We examined Pavlovian-instrumental transfer (PIT) by measuring the ability of an auditory cue associated with food reward to promote an instrumental (lever press) response. In a separate experiment, we assessed the acquisition and extinction of a simple stimulus-reward instrumental behavior on a touch screen based task. Reinstatement of this behavior was then examined following either continuous exposure to cues (conditioned reinforcers, CRs) associated with reward, brief reward and CR exposure, or brief reward exposure followed by continuous CR exposure. The third paradigm examined sensitivity of an instrumental (lever press) response to devaluation of food reward (a probe for outcome insensitive, habitual behavior) by repeated pairing with malaise. Results showed that C57BL/6J mice displayed robust PIT, as well as clear extinction and reinstatement, but were insensitive to reinforcer devaluation. DBA/2J mice showed good PIT and (rewarded) reinstatement, but were slow to extinguish and did not show reinforcer devaluation or significant CR-reinstatement. BALB/cJ mice also displayed good PIT, extinction and reinstatement, and retained instrumental responding following devaluation, but, unlike the other strains, demonstrated reduced Pavlovian approach behavior (food magazine head entries). Overall, these assays provide robust paradigms for future studies using the mouse to elucidate the neural, molecular and genetic factors underpinning reward-related behaviors relevant to addiction research.

Marker assisted speed congenics technique is commonly used to facilitate backcrossing of mouse strains in nearly half the time it normally takes otherwise. Traditionally, the technique is performed by analyzing PCR amplified regions of simple sequence length polymorphism (SSLP) markers between the recipient and donor strains: offspring with the highest number of markers showing the recipient genome across all chromosomes is chosen for the next generation. Although there are well-defined panels of SSLP makers established between certain pairs of mice strains, they are incomplete for most strains. The availability of well-established marker sets for speed congenic screens would enable the scientific community to transfer mutations across strain backgrounds. In this study, we tested the suitability of over 400 SSLP marker sets among 10 mouse strains commonly used for generating genetically engineered models. The panel of markers presented here can readily identify the specified strains and will be quite useful in marker assisted speed congenic screens. Moreover, unlike newer single nucleotide polymorphism (SNP) array methods which require sophisticated equipment, the SSLP markers panel described here only uses PCR and agarose gel electrophoresis of amplified products; therefore it can be performed in most research laboratories.

Due in part to their rich behavioral repertoire rats have been widely used in behavioral studies of drug abuse-related traits for decades. However, the mouse became the model of choice for researchers exploring the genetic underpinnings of addiction after the first mouse study was published demonstrating the capability of engineering the mouse genome through embryonic stem cell technology. The sequencing of the mouse genome and more recent re-sequencing of numerous inbred mouse strains have further cemented the status of mice as the premier mammalian organism for genetic studies. As a result, many of the behavioral paradigms initially developed and optimized for rats have been adapted to mice. However, numerous complex and interesting drug abuse-related behaviors that can be studied in rats are very difficult or impossible to adapt for use in mice, impeding the genetic dissection of those traits. Now, technological advances have removed many of the historical limitations of genetic studies in rats. For instance, the rat genome has been sequenced and many inbred rat strains are now being re-sequenced and outbred rat stocks are being used to fine-map QTLs. In addition, it is now possible to create "knockout" rats using zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs) and related techniques. Thus, rats can now be used to perform quantitative genetic studies of sophisticated behaviors that have been difficult or impossible to study in mice. This article is part of a Special Issue entitled 'NIDA 40th Anniversary Issue'.

The human pathogen L. monocytogenes and the animal pathogen L. ivanovii, together with four other species isolated from symptom-free animals, form the "Listeria sensu stricto" clade. The members of the second clade, "Listeria sensu lato", are believed to be solely environmental bacteria without the ability to colonize mammalian hosts. To identify novel determinants that contribute to infection by L. monocytogenes, the causative agent of the foodborne disease listeriosis, we performed a genome comparison of the two clades and found 151 candidate genes that are conserved in the Listeria sensu stricto species. Two factors were investigated further in vitro and in vivo. A mutant lacking an ATP-binding cassette transporter exhibited defective adhesion and invasion of human Caco-2 cells. Using a mouse model of foodborne L. monocytogenes infection, a reduced number of the mutant strain compared to the parental strain was observed in the small intestine and the liver. Another mutant with a defective 1,2-propanediol degradation pathway showed reduced persistence in the stool of infected mice, suggesting a role of 1,2-propanediol as a carbon and energy source of listeriae during infection. These findings reveal the relevance of novel factors for the colonization process of L. monocytogenes.

Oleuropein, the major compound found in olive leaves, has been reported to exert numerous pharmacological properties, including anti-inflammatory, anti-diabetic and anti-cancer effects. The purpose of this study was to evaluate, for the first time, the effect of oleuropein-rich leaf extracts (ORLE) in already-developed colon tumours arising in Apc (adenomatous polyposis coli) mutated PIRC rats (F344/NTac-Apcam1137). Here, we were able to investigate in parallel the anti-cancer effect of ORLE, both in vivo and in vitro, and its anti-inflammatory effect on macrophages, representing a critical and abundant population in most solid tumour microenvironment. We found that in vivo ORLE treatment promoted apoptosis and attenuated iNOS activity both in colon tumours as in peritoneal macrophages of PIRC rats. We this confirmed in vitro using primary RAW264.7 cells: ORLE reduced iNOS activity in parallel with COX-2 and pro-inflammatory cytokines, such as IL-1β, IL-6 and TGF-β. These findings suggest that ORLE possess a strong anti-inflammatory activity, which could be crucial for dampening the pro-tumourigenic activity elicited by a chronic inflammatory state generated by either tumour cells or tumour-associated macrophages.

Neurodegenerative diseases commonly involve the disruption of circadian rhythms. Studies indicate that mutant Huntingtin (mHtt), the cause of Huntington's disease (HD), disrupts circadian rhythms often before motor symptoms are evident. Yet little is known about the molecular mechanisms by which mHtt impairs circadian rhythmicity and whether circadian clocks can modulate HD pathogenesis. To address this question, we used a Drosophila HD model. We found that both environmental and genetic perturbations of the circadian clock alter mHtt-mediated neurodegeneration. To identify potential genetic pathways that mediate these effects, we applied a behavioral platform to screen for clock-regulated HD suppressors, identifying a role for Heat Shock Protein 70/90 Organizing Protein (Hop). Hop knockdown paradoxically reduces mHtt aggregation and toxicity. These studies demonstrate a role for the circadian clock in a neurodegenerative disease model and reveal a clock-regulated molecular and cellular pathway that links clock function to neurodegenerative disease.

The S334ter rhodopsin (Rho) rat (line 4) bears the rhodopsin gene with an early termination codon at residue 334 that is a model for several such mutations found in human patients with autosomal dominant retinitis pigmentosa (ADRP). The Unfolded Protein Response (UPR) is implicated in the pathophysiology of several retinal disorders including ADRP in P23H Rho rats. The aim of this study was to examine the onset of UPR gene expression in S334ter Rho retinas to determine if UPR is activated in ADRP animal models and to investigate how the activation of UPR molecules leads to the final demise of S334ter Rho photoreceptors. RT-PCR was performed to evaluate the gene expression profiles for the P10, P12, P15, and P21 stages of the development and progression of ADRP in S334ter Rho photoreceptors. We determined that during the P12-P15 period, ER stress-related genes are strongly upregulated in transgenic retinas, resulting in the activation of the UPR that was confirmed using western blot analysis and RT-PCR. The activation of UPR was associated with the increased expression of JNK, Bik, Bim, Bid, Noxa, and Puma genes and cleavage of caspase-12 that together with activated calpains presumably compromise the integrity of the mitochondrial MPTP, leading to the release of pro-apoptotic AIF1 into the cytosol of S334ter Rho photoreceptor cells. Therefore, two major cross-talking pathways, the UPR and mitochondrial MPTP occur in S334ter-4 Rho retina concomitantly and eventually promote the death of the photoreceptor cells.

Through mutant selection on agar containing pyrazinoic acid (POA), the bioactive form of the prodrug pyrazinamide (PZA), we recently showed that missense mutations in the aspartate decarboxylase PanD and the unfoldase ClpC1, and loss-of-function mutation of polyketide synthases Mas and PpsA-E involved in phthiocerol dimycocerosate synthesis, cause resistance to POA and PZA in Mycobacterium tuberculosis. Here we first asked whether these in vitro-selected POA/PZA-resistant mutants are attenuated in vivo, to potentially explain the lack of evidence of these mutations among PZA-resistant clinical isolates. Infection of mice with panD, clpC1, and mas/ppsA-E mutants showed that whereas growth of clpC1 and mas/ppsA-E mutants was attenuated, the panD mutant grew as well as the wild-type. To determine whether these resistance mechanisms can emerge within the host, mice infected with wild-type M. tuberculosis were treated with POA, and POA-resistant colonies were confirmed for PZA and POA resistance. Genome sequencing revealed that 82 and 18% of the strains contained missense mutations in panD and clpC1, respectively. Consistent with their lower fitness and POA resistance level, independent mas/ppsA-E mutants were not found. In conclusion, we show that the POA/PZA resistance mechanisms due to panD and clpC1 missense mutations are recapitulated in vivo. Whereas the representative clpC1 mutant was attenuated for growth in the mouse infection model, providing a possible explanation for their absence among clinical isolates, the growth kinetics of the representative panD mutant was unaffected. Why POA/PZA resistance-conferring panD mutations are observed in POA-treated mice but not yet among clinical strains isolated from PZA-treated patients remains to be determined.