RESUMEN
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.
Asunto(s)
Retrovirus Endógenos , Variación Genética , Genoma/genética , Ratas Endogámicas/genética , Ratas Endogámicas/virología , Proteínas del Envoltorio Viral/genética , Animales , Elementos Transponibles de ADN/genética , Retrovirus Endógenos/clasificación , Retrovirus Endógenos/genética , Femenino , Genes Virales/genética , Masculino , Ratones , Datos de Secuencia Molecular , Ratas , Análisis de Secuencia de ADN , Especificidad de la Especie , Integración ViralRESUMEN
Investigation of proteinuria, whose pathophysiology remains incompletely understood, is confounded by differences in the phenotype between males and females. We initiated a sex-specific geno-transcriptomic dissection of proteinuria in uninephrectomized male and female Sabra rats that spontaneously develop focal and segmental glomerulosclerosis, testing the hypothesis that different mechanisms might underlie the pathophysiology of proteinuria between the sexes. In the genomic arm, we scanned the genome of 136 male and 111 female uninephrectomized F2 populations derived from crosses between SBH/y and SBN/y. In males, we identified proteinuria-related quantitative trait loci (QTLs) on RNO2 and 20 and protective QTLs on RNO6 and 9. In females, we detected proteinuria-related QTLs on RNO11, 13, and 20. The only QTL overlap between the sexes was on RNO20. Using consomic strains, we confirmed the functional significance of this QTL in both sexes. In the transcriptomic arm, we searched on a genomewide scale for genes that were differentially expressed in kidneys of SBH/y and SBN/y with and without uninephrectomy. These studies identified within each sex differentially expressed genes of relevance to proteinuria. Integrating genomics with transcriptomics, we identified differentially expressed genes that mapped within the boundaries of the proteinuria-related QTLs, singling out 24 transcripts in males and 30 in females, only 4 of which (Tubb5, Ubd, Psmb8, and C2) were common to both sexes. Data mining revealed that these transcripts are involved in multiple molecular mechanisms, including immunity, inflammation, apoptosis, matrix deposition, and protease activity, with no single molecular pathway predominating in either sex. These results suggest that the pathophysiology of proteinuria is highly complex and that some of the underlying mechanisms are shared between the sexes, while others are sex specific and may account for the difference in the proteinuric phenotype between males and females.
Asunto(s)
Perfilación de la Expresión Génica , Nefrectomía , Proteinuria/genética , Caracteres Sexuales , Animales , Análisis por Conglomerados , Femenino , Masculino , Fenotipo , Sitios de Carácter Cuantitativo , RatasRESUMEN
The hypodactylous (hd) locus impairs limb development and spermatogenesis, leading to male infertility in rats. We show that the hd mutation is caused by an insertion of an endogenous retrovirus into intron 10 of the Cntrob gene. The retroviral insertion in hd mutant rats disrupts the normal splicing of Cntrob transcripts and results in the expression of a truncated protein. During the final phase of spermiogenesis, centrobin localizes to the manchette, centrosome, and the marginal ring of the spermatid acroplaxome, where it interacts with keratin 5-containing intermediate filaments. Mutant spermatids show a defective acroplaxome marginal ring and separation of the centrosome from its normal attachment site of the nucleus. This separation correlates with a disruption of head-tail coupling apparatus, leading to spermatid decapitation during the final step of spermiogenesis and the absence of sperm in the epididymis. Cntrob may represent a novel candidate gene for presently unexplained hereditary forms of teratozoospermia and the "easily decapitated sperm syndrome" in humans.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Genes Homeobox/genética , Proteínas de Homeodominio/genética , Cabeza del Espermatozoide/metabolismo , Cola del Espermatozoide/metabolismo , Espermatogénesis/genética , Animales , Far-Western Blotting , Centrosoma/metabolismo , Retrovirus Endógenos/genética , Epidídimo/metabolismo , Técnica del Anticuerpo Fluorescente , Proteínas de Homeodominio/metabolismo , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Intrones/genética , Queratina-5/genética , Queratina-5/metabolismo , Masculino , Microscopía Electrónica , Mutación/genética , Transporte de Proteínas/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espermátides/metabolismoRESUMEN
Genetic alterations can unpredictably compromise the wellbeing of animals. Thus, more or less harmful phenotypes might appear in the animals used in research projects even when they are not subjected to experimental treatments. The severity classification of suffering has become an important issue since the implementation of Directive 2010/63/EU on the protection of animals used for scientific purposes. Accordingly, the breeding and maintenance of genetically altered (GA) animals which are likely to develop a harmful phenotype has to be authorized. However, a determination of the degree of severity is rather challenging due to the large variety of phenotypes. Here, the Working Group of Berlin Animal Welfare Officers (WG Berlin AWO) provides field-tested guidelines on severity assessment and classification of GA rodents. With a focus on basic welfare assessment and severity classification we provide a list of symptoms that have been classified as non-harmful, mild, moderate or severe burdens. Corresponding monitoring and refinement strategies as well as specific housing requirements have been compiled and are strongly recommended to improve hitherto applied breeding procedures and conditions. The document serves as a guide to determine the degree of severity for an observed phenotype. The aim is to support scientists, animal care takers, animal welfare bodies and competent authorities with this task, and thereby make an important contribution to a European harmonization of severity assessments for the continually increasing number of GA rodents.
Asunto(s)
Bienestar del Animal/normas , Cruzamiento , Ratones , Fenotipo , Ratas , Animales , Animales de Laboratorio , Unión EuropeaRESUMEN
BACKGROUND: Heterochromatin has been reported to be a major silencing compartment during development and differentiation. Prominent heterochromatin compartments are located at the nuclear periphery and inside the nucleus (e.g., pericentric heterochromatin). Whether the position of a gene in relation to some or all heterochromatin compartments matters remains a matter of debate, which we have addressed in this study. Answering this question demanded solving the technical challenges of 3D measurements and the large-scale morphological changes accompanying cellular differentiation. RESULTS: Here, we investigated the proximity effects of the nuclear periphery and pericentric heterochromatin on gene expression and additionally considered the effect of neighboring genomic features on a gene's nuclear position. Using a well-established myogenic in vitro differentiation system and a differentiation-independent heterochromatin remodeling system dependent on ectopic MeCP2 expression, we first identified genes with statistically significant expression changes by transcriptional profiling. We identified nuclear gene positions by 3D fluorescence in situ hybridization followed by 3D distance measurements toward constitutive and facultative heterochromatin domains. Single-cell-based normalization enabled us to acquire morphologically unbiased data and we finally correlated changes in gene positioning to changes in transcriptional profiles. We found no significant correlation of gene silencing and proximity to constitutive heterochromatin and a rather unexpected inverse correlation of gene activity and position relative to facultative heterochromatin at the nuclear periphery. CONCLUSION: In summary, our data question the hypothesis of heterochromatin as a general silencing compartment. Nonetheless, compared to a simulated random distribution, we found that genes are not randomly located within the nucleus. An analysis of neighboring genomic context revealed that gene location within the nucleus is rather dependent on CpG islands, GC content, gene density, and short and long interspersed nuclear elements, collectively known as RIDGE (regions of increased gene expression) properties. Although genes do not move away/to the heterochromatin upon up-/down-regulation, genomic regions with RIDGE properties are generally excluded from peripheral heterochromatin. Hence, we suggest that individual gene activity does not influence gene positioning, but rather chromosomal context matters for sub-nuclear location.
RESUMEN
Rat hypodactyly (hd) mutation is characterized by abnormal spermatogenesis and sperm decapitation, limb malformation (missing digits II and III) and growth retardation. We have previously reported centrobin (centrosome BRCA2-interacting protein) truncation at the C-terminus in the hd mutant. Here, we report data from a transgenic rescue experiment carried out to determine a role of centrobin in pathogenesis of hd. The transgenic construct, consisting of full-length-coding cDNA linked to a ubiquitous strong promoter/enhancer combination, was inserted to chromosome 16 into a LINE repeat. No known gene is present in the vicinity of the insertion site. Transgenic centrobin was expressed in all tissues tested, including testis. Transgenic animals show normal body weight and limb morphology as well as average weight of testis and epididymis. Yet, abnormal spermatogenesis and sperm decapitation persisted in the transgenic animals. Western blotting showed the coexistence of full-length and truncated or partially degraded centrobin in sperm of the rescued transgenic animals. Immunocytochemistry showed a buildup of centrobin and ODF2 (outer dense fiber 2) at the sperm decapitation site in the hd mutant and rescued transgenic rats. Additional findings included bulge-like formations and thread-like focal dissociations along the sperm flagellum and the organization of multiple whorls of truncated sperm flagella in the epididymal lumen. We conclude that centrobin is essential for normal patterning of the limb autopod. Centrobin may be required for stabilizing the attachment of the sperm head to flagellum and for maintaining the structural integrity of the sperm flagellum. We postulate that the presence of truncated centrobin, coexisting with full-length centrobin, together with incorrect timing of transgenic centrobin expression may hamper the rescue of fertility in hd male rats.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Homeodominio/genética , Deformidades Congénitas de las Extremidades/genética , Mutación , Animales , Proteínas de Ciclo Celular/metabolismo , Epidídimo/patología , Fertilidad/genética , Expresión Génica , Proteínas de Choque Térmico/metabolismo , Masculino , Ratones , Tamaño de los Órganos/genética , Transporte de Proteínas , Ratas , Ratas Transgénicas , Espermatozoides/crecimiento & desarrollo , Espermatozoides/metabolismo , Testículo/patologíaRESUMEN
Cerebral ischemic small vessel disease (SVD) is the leading cause of vascular dementia and a major contributor to stroke in humans. Dominant mutations in NOTCH3 cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a genetic archetype of cerebral ischemic SVD. Progress toward understanding the pathogenesis of this disease and developing effective therapies has been hampered by the lack of a good animal model. Here, we report the development of a mouse model for CADASIL via the introduction of a CADASIL-causing Notch3 point mutation into a large P1-derived artificial chromosome (PAC). In vivo expression of the mutated PAC transgene in the mouse reproduced the endogenous Notch3 expression pattern and main pathological features of CADASIL, including Notch3 extracellular domain aggregates and granular osmiophilic material (GOM) deposits in brain vessels, progressive white matter damage, and reduced cerebral blood flow. Mutant mice displayed attenuated myogenic responses and reduced caliber of brain arteries as well as impaired cerebrovascular autoregulation and functional hyperemia. Further, we identified a substantial reduction of white matter capillary density. These neuropathological changes occurred in the absence of either histologically detectable alterations in cerebral artery structure or blood-brain barrier breakdown. These studies provide in vivo evidence for cerebrovascular dysfunction and microcirculatory failure as key contributors to hypoperfusion and white matter damage in this genetic model of ischemic SVD.
Asunto(s)
Isquemia Encefálica/genética , Circulación Cerebrovascular/genética , Animales , Vasos Sanguíneos/patología , Barrera Hematoencefálica , Isquemia Encefálica/patología , CADASIL/genética , CADASIL/patología , Arterias Cerebrales/patología , Cromosomas Artificiales/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Homeostasis , Humanos , Ratones , Ratones Transgénicos , Receptor Notch3 , Receptores Notch/genéticaAsunto(s)
Secuencias Repetitivas Esparcidas , Reacción en Cadena de la Polimerasa/métodos , Animales , Secuencia de Bases , Cromosomas Artificiales Bacterianos/genética , Cromosomas Artificiales de Bacteriófagos P1/genética , Cromosomas Artificiales de Levadura/genética , Cartilla de ADN/genética , Sondas de ADN , Electroforesis en Gel de Campo Pulsado , Biblioteca Genómica , Humanos , Hibridación de Ácido NucleicoRESUMEN
We aimed to identify genetic variants associated with heart failure by using a rat model of the human disease. We performed invasive cardiac hemodynamic measurements in F2 crosses between spontaneously hypertensive heart failure (SHHF) rats and reference strains. We combined linkage analyses with genome-wide expression profiling and identified Ephx2 as a heart failure susceptibility gene in SHHF rats. Specifically, we found that cis variation at Ephx2 segregated with heart failure and with increased transcript expression, protein expression and enzyme activity, leading to a more rapid hydrolysis of cardioprotective epoxyeicosatrienoic acids. To confirm our results, we tested the role of Ephx2 in heart failure using knockout mice. Ephx2 gene ablation protected from pressure overload-induced heart failure and cardiac arrhythmias. We further demonstrated differential regulation of EPHX2 in human heart failure, suggesting a cross-species role for Ephx2 in this complex disease.
Asunto(s)
Modelos Animales de Enfermedad , Epóxido Hidrolasas/genética , Predisposición Genética a la Enfermedad , Insuficiencia Cardíaca/genética , Ratas/genética , Animales , Mapeo Cromosómico , Epóxido Hidrolasas/análisis , Epóxido Hidrolasas/metabolismo , Perfilación de la Expresión Génica , Ligamiento Genético , Insuficiencia Cardíaca/enzimología , Insuficiencia Cardíaca/fisiopatología , Humanos , Hipertensión/complicaciones , Hipertensión/genética , Ratones , Ratones Noqueados , Miocardio/enzimología , Polimorfismo Genético , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Sitios de Carácter Cuantitativo , Ratas Mutantes , Análisis de Secuencia de ADN , Eliminación de Secuencia , Factor de Transcripción AP-1/metabolismoRESUMEN
Evidence for blood pressure quantitative trait loci (QTLs) on rat chromosome 10 has been found in multiple independent studies. Analysis of the homologous region on human chromosome 17 revealed significant linkage to blood pressure. The critical segment on human chromosome 17 spans a large interval containing the genes Itga2b, Gfap, and Itgb3. Therefore, findings in the rat may help to refine the position of blood pressure-regulating loci, assuming a common molecular cause across species. However, it has recently been suggested that the gene order in human, rat, and mouse is not conserved in this region, leaving uncertainty about the overlap of the blood pressure- regulating region between human chromosome 17 and rat chromosome 10. We have performed a detailed comparative analysis among human, mouse, and rat, defining the segment in question, by obtaining gene structure information in silico and by radiation hybrid mapping. It is of interest that this region also contains Wnk4, a gene previously identified to cause pseudohypoaldosteronism type II and human hypertension. Our results definitively show that the conserved synteny extends among human chromosome 17, rat chromosome 10, and mouse chromosome 11, demonstrating an overlap between previously localized blood pressure QTLs in humans and rats.
Asunto(s)
Presión Sanguínea/genética , Cromosomas Humanos Par 17 , Carácter Cuantitativo Heredable , Animales , Humanos , Ratones , Proteínas Serina-Treonina Quinasas/genética , Mapeo de Híbrido por Radiación , Ratas , SinteníaRESUMEN
The fatty acid binding protein 6 gene (Fabp6) codes for ileal lipid binding protein. After sequencing of rat Fabp6, the gene was localized in a radiation hybrid (RH) map on chromosome 10. An intronless Fabp6 segment was found in four related rat inbred strains (SHR; SHRSP; WKY; and OKA), but not in 62 other rat inbred strains. The intronless Fabp6 segment, which might be a pseudogene of Fabp6, was localized on rat chromosome 15.
Asunto(s)
Proteínas Portadoras/genética , Cromosomas de los Mamíferos/genética , Intrones/genética , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Exones/genética , Proteínas de Unión a Ácidos Grasos , Hormonas Gastrointestinales , Datos de Secuencia Molecular , Seudogenes/genética , Ratas , Ratas Endogámicas , Análisis de Secuencia de ADNRESUMEN
As part of the effort to sequence the genome of Rattus norvegicus, we constructed a physical map comprised of fingerprinted bacterial artificial chromosome (BAC) clones from the CHORI-230 BAC library. These BAC clones provide approximately 13-fold redundant coverage of the genome and have been assembled into 376 fingerprint contigs. A yeast artificial chromosome (YAC) map was also constructed and aligned with the BAC map via fingerprinted BAC and P1 artificial chromosome clones (PACs) sharing interspersed repetitive sequence markers with the YAC-based physical map. We have annotated 95% of the fingerprint map clones in contigs with coordinates on the version 3.1 rat genome sequence assembly, using BAC-end sequences and in silico mapping methods. These coordinates have allowed anchoring 358 of the 376 fingerprint map contigs onto the sequence assembly. Of these, 324 contigs are anchored to rat genome sequences localized to chromosomes, and 34 contigs are anchored to unlocalized portions of the rat sequence assembly. The remaining 18 contigs, containing 54 clones, still require placement. The fingerprint map is a high-resolution integrative data resource that provides genome-ordered associations among BAC, YAC, and PAC clones and the assembled sequence of the rat genome.