RESUMEN
Positional cloning of hereditary deafness genes is a direct approach to identify molecules and mechanisms underlying auditory function. Here we report a locus for dominant deafness, DFNA36, which maps to human chromosome 9q13-21 in a region overlapping the DFNB7/B11 locus for recessive deafness. We identified eight mutations in a new gene, transmembrane cochlear-expressed gene 1 (TMC1), in a DFNA36 family and eleven DFNB7/B11 families. We detected a 1.6-kb genomic deletion encompassing exon 14 of Tmc1 in the recessive deafness (dn) mouse mutant, which lacks auditory responses and has hair-cell degeneration. TMC1 and TMC2 on chromosome 20p13 are members of a gene family predicted to encode transmembrane proteins. Tmc1 mRNA is expressed in hair cells of the postnatal mouse cochlea and vestibular end organs and is required for normal function of cochlear hair cells.
Asunto(s)
Sordera/genética , Genes Dominantes , Genes Recesivos , Células Ciliadas Auditivas/fisiopatología , Mutación , Alelos , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , Cromosomas Humanos Par 9 , Femenino , Humanos , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Familia de Multigenes , Linaje , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de AminoácidoRESUMEN
Mouse knockout technology provides a powerful means of elucidating gene function in vivo, and a publicly available genome-wide collection of mouse knockouts would be significantly enabling for biomedical discovery. To date, published knockouts exist for only about 10% of mouse genes. Furthermore, many of these are limited in utility because they have not been made or phenotyped in standardized ways, and many are not freely available to researchers. It is time to harness new technologies and efficiencies of production to mount a high-throughput international effort to produce and phenotype knockouts for all mouse genes, and place these resources into the public domain.
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Ratones Noqueados , Creación de Embriones para Investigación , Alelos , Animales , Investigación Genética , Ratones , Fenotipo , Creación de Embriones para Investigación/economíaRESUMEN
In 2007, the International Knockout Mouse Consortium (IKMC) made the ambitious promise to generate mutations in virtually every protein-coding gene of the mouse genome in a concerted worldwide action. Now, 5 years later, the IKMC members have developed high-throughput gene trapping and, in particular, gene-targeting pipelines and generated more than 17,400 mutant murine embryonic stem (ES) cell clones and more than 1,700 mutant mouse strains, most of them conditional. A common IKMC web portal (www.knockoutmouse.org) has been established, allowing easy access to this unparalleled biological resource. The IKMC materials considerably enhance functional gene annotation of the mammalian genome and will have a major impact on future biomedical research.
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Ratones Noqueados/genética , Animales , Internacionalidad , Internet , RatonesRESUMEN
We, the directors of the 27 NIH institutes and centers, wanted to respond to the points made by Andrew Marks in his recent editorial. While we appreciate that the scientific community has concerns, the current initiatives and directions of the NIH have been developed through planning processes that reflect openness and continued constituency input, all aimed at assessing scientific opportunities and addressing public health needs.
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National Institutes of Health (U.S.) , Política Organizacional , Humanos , National Institutes of Health (U.S.)/economía , National Institutes of Health (U.S.)/organización & administración , Estados UnidosRESUMEN
Mice with a targeted disruption of bombesin receptor subtype-3 (BRS-3 KO) develop hyperphagia, obesity, hypertension, and impaired glucose metabolism. However, the factors contributing to their phenotype have not been clearly established. To determine whether their obesity is a result of increased food intake or a defect in energy regulation, we matched the caloric intake of BRS-3 KO mice to wild-type (WT) ad libitum (ad lib)-fed controls over 21 wk. Although BRS-3 KO ad lib-fed mice were 29% heavier, the body weights of BRS-3 KO pair-fed mice did not differ from WT ad lib-fed mice. Pair-feeding BRS-3 KO mice normalized plasma insulin but failed to completely reverse increased adiposity and leptin levels. Hyperphagia in ad lib-fed KO mice was due to an increase in meal size without a compensatory decrease in meal frequency resulting in an increase in total daily food intake. An examination of neuropeptide Y, proopiomelanocortin, and agouti-related peptide gene expression in the arcuate nucleus revealed that BRS-3 KO mice have some deficits in their response to energy regulatory signals. An evaluation of the satiety effects of cholecystokinin, bombesin, and gastrin-releasing peptide found no differences in feeding suppression by these peptides. We conclude that hyperphagia is a major factor leading to increased body weight and hyperinsulinemia in BRS-3 KO mice. However, our finding that pair-feeding did not completely normalize fat distribution and plasma leptin levels suggests there is also a metabolic dysregulation that may contribute to, or sustain, their obese phenotype.
Asunto(s)
Hiperfagia/complicaciones , Hiperfagia/metabolismo , Obesidad/etiología , Obesidad/metabolismo , Receptores de Bombesina/metabolismo , Adiposidad/efectos de los fármacos , Adiposidad/fisiología , Animales , Peso Corporal/efectos de los fármacos , Peso Corporal/fisiología , Bombesina/farmacología , Colecistoquinina/farmacología , Ingestión de Alimentos/efectos de los fármacos , Ingestión de Alimentos/fisiología , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/fisiología , Péptido Liberador de Gastrina/farmacología , Glucosa/metabolismo , Hiperinsulinismo/etiología , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Insulina/sangre , Leptina/sangre , Masculino , Ratones , Ratones Noqueados , Saciedad/fisiología , Aumento de PesoRESUMEN
PURPOSE: To determine whether cadherin 23 and protocadherin 15 can substitute for one another in the maintenance of the retina and other tissues in the mouse. Does homozygosity for both v and av mutant alleles (i.e., a double homozygous mouse) cause retinal degeneration or an obvious retinal histopathology? METHODS: We generated mice homozygous for both Cdh23(v-6J) and Pcdh15(av-Jfb) alleles. The retinal phenotypes of double heterozygous and double homozygous mutant mice were determined by light microscopy and electroretinography (ERG). Histology on 32 different tissues, scanning electron microscopy of organ of Corti hair cells as well as serum biochemical and hematological examinations were evaluated. RESULTS: ERG waves of double heterozygous and double homozygous mice showed similar shape, growth of the amplitude with intensity, and implicit time for both rod and cone pathway mediated responses. Mice homozygous for both Cdh23(v-6J) and Pcdh15(av-Jfb) mutations showed no sign of retinitis pigmentosa or photoreceptor degeneration but, as expected, were deaf and had disorganized hair cell sensory bundles. CONCLUSIONS: The simultaneous presence of homozygous mutant alleles of cadherin 23 and protocadherin 15 results only in deafness, not retinal degeneration or any other additional obvious phenotype of the major organ systems. We conclude that in the mouse cadherin 23 or protocadherin 15 appear not to compensate for one another to maintain the retina.
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Alelos , Homocigoto , Degeneración Retiniana/genética , Empalme Alternativo , Animales , Proteínas Relacionadas con las Cadherinas , Cadherinas/genética , Núcleo Celular/patología , Cilios/ultraestructura , Electrorretinografía , Ojo/patología , Ojo/ultraestructura , Heterocigoto , Ratones , Ratones Mutantes , Fenotipo , Precursores de Proteínas/genéticaRESUMEN
The T2Rs belong to a multi-gene family of G-protein-coupled receptors responsible for the detection of ingested bitter-tasting compounds. The T2Rs are conserved among mammals with the human and mouse gene families consisting of about 25 members. In the present study we address the signalling properties of human and mouse T2Rs using an in vitro reconstitution system in which both the ligands and G-proteins being assayed can be manipulated independently and quantitatively assessed. We confirm that the mT2R5, hT2R43 and hT2R47 receptors respond selectively to micromolar concentrations of cycloheximide, aristolochic acid and denatonium respectively. We also demonstrate that hT2R14 is a receptor for aristolochic acid and report the first characterization of the ligand specificities of hT2R7, which is a broadly tuned receptor responding to strychnine, quinacrine, chloroquine and papaverine. Using these defined ligand-receptor interactions, we assayed the ability of the ligand-activated T2Rs to catalyse GTP binding on divergent members of the G(alpha) family including three members of the G(alphai) subfamily (transducin, G(alphai1) and G(alphao)) as well as G(alphas) and G(alphaq). The T2Rs coupled with each of the three G(alphai) members tested. However, none of the T2Rs coupled to either G(alphas) or G(alphaq), suggesting the T2Rs signal primarily through G(alphai)-mediated signal transduction pathways. Furthermore, we observed different G-protein selectivities among the T2Rs with respect to both G(alphai) subunits and G(betagamma) dimers, suggesting that bitter taste is transduced by multiple G-proteins that may differ among the T2Rs.
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Receptores Acoplados a Proteínas G/fisiología , Gusto/fisiología , Animales , Ácidos Aristolóquicos/metabolismo , Cicloheximida/metabolismo , Humanos , Ratones , Compuestos de Amonio Cuaternario/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Transducina/metabolismoRESUMEN
PURPOSE: Mutations of PCDH15, the gene encoding protocadherin 15, cause either nonsyndromic deafness DFNB23 or Usher syndrome type 1F (USH1F) in humans and deafness with balance problems in Ames waltzer (av) mice. Persons with USH1 usually begin to exhibit signs of retinitis pigmentosa (RP) in early adolescence, but av mice are reported to have functional retinas. In this study, the auditory, visual and molecular biological phenotype of Pcdh15av-5J and Pcdh15av-Jfb mice is characterized, and their usefulness as animal models of USH1 is evaluated. METHODS: Hearing thresholds of mice between 6 and 10 weeks of age were measured by auditory brain stem response (ABR). Immunohistochemistry and histology were used to examine the effect of homozygosity of Pcdh15av-5J on stereocilia bundles of inner ear hair cells and on the photoreceptor cells of the retina. Scotopic and photopic Ganzfeld ERGs were recorded from homozygous Pcdh15av-5J and Pcdh15av-Jfb mice at different ages. Heterozygous littermates served as control subjects. Measurements of the width of the outer nuclear layer (ONL) and the length of rod photoreceptor outer segment (ROS) were made. RESULTS: Homozygous Pcdh15av-5J mice have profound hearing loss and disorganized stereocilia bundles of inner ear hair cells. Compared with heterozygous littermates, homozygous Pcdh15av-5J and Pcdh15av-Jfb mutant mice had scotopic ERG amplitudes consistently reduced by approximately 40% at all light intensities. The b-to-a-wave ratio confirmed that the a- and b-waves were reduced proportionally in homozygous mutant mice. Histologic measurements of retinal sections revealed no significant differences in either the ONL width or the ROS length as a function of genotype. The protocadherin 15 labeling pattern with antisera PB303 in the retina of both heterozygous and homozygous Pcdh15av-5J mice was indistinguishable from the wild type. Wild-type Pcdh15 have many alternatively spliced isoforms. A novel isoform was found in the retina of homozygous Pcdh15av-5J mice, which appears to circumvent the effect of the mutant allele (IVS14-2A-->G), which causes skipping of exon 14, a shift in the translation reading frame and a premature stop codon in exon 15. CONCLUSIONS: Pcdh15(av-5J) and Pcdh15(av-Jfb) mice do not faithfully mimic the RP found in USH1 due to mutations of PCDH15, but have significantly attenuated ERG function in the absence of histologic change. The decline in ERG amplitude with a preserved b-to-a-wave ratio suggests a role for Pcdh15 in retinal function and/or generation of the ERG potentials. Understanding the molecular mechanism by which av mice circumvent degeneration of the retina might offer insights into potential therapies for USH1.
Asunto(s)
Empalme Alternativo/genética , Cadherinas/genética , Sordera/congénito , Precursores de Proteínas/genética , Retina/fisiopatología , Retinitis Pigmentosa/genética , Enfermedades Vestibulares/genética , Animales , Umbral Auditivo , Proteínas Relacionadas con las Cadherinas , Modelos Animales de Enfermedad , Electrorretinografía , Potenciales Evocados Auditivos del Tronco Encefálico , Femenino , Células Ciliadas Auditivas Internas/metabolismo , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Mutación , Fenotipo , Células Fotorreceptoras de Vertebrados/metabolismo , Retinitis Pigmentosa/fisiopatología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Recent studies have shown that aberrantly expressed gastrin-releasing peptide (GRP) and its receptor (GRP-R) critically regulate tumor cell differentiation in colon cancers developing in humans and mice. This finding suggested that the ability of GRP/GRP-R to promote a well-differentiated phenotype in colon cancer might reflect a re-capitulation of a normal role in regulating intestinal organogenesis. To determine if this was the case, we compared and contrasted intestinal development in GRPR-/- mice with their wild type littermates. GRP/GRP-R co-expression in wild type mice was only observed in villous enterocytes between N-1 and N-12. During this time frame villous growth was completely attenuated in GRPR-/- mice. The contribution of GRP/GRP-R to villous growth was due to their act in increasing enterocyte proliferation prior to N-8 but increasing enterocyte size thereafter. From N-12 onwards, small intestinal villous growth in GRPR-/- mice resumed such that no difference in this structure could be detected at adulthood between mice of either genotype. We next studied GRP/GRP-R expression in human abortuses. These proteins were co-expressed by villous enterocytes only between weeks 14 and 20 post-conception, a time frame analogous to when they are expressed in the murine intestine. Thus, this study shows for the first time that GRP/GRP-R play a transient and non-critical role in intestinal development, yet provides a rationale for their re-appearance in colon cancer.
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Sistema Digestivo/embriología , Enterocitos/metabolismo , Péptido Liberador de Gastrina/fisiología , Receptores de Bombesina/fisiología , Feto Abortado , Animales , División Celular , Separación Celular , Citometría de Flujo , Péptido Liberador de Gastrina/biosíntesis , Genotipo , Humanos , Inmunohistoquímica , Ratones , Fenotipo , Receptores de Bombesina/biosíntesis , Factores de TiempoRESUMEN
Human embryonic stem cell research offers the promise to elucidate some of the molecular mechanisms that underlie differentiation into specialized types. This knowledge may someday be used to develop new treatments for cellular degenerative diseases. National Institutes of Health has taken several steps to expedite progress in this new field.
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Investigaciones con Embriones , Células Madre Embrionarias/citología , National Institutes of Health (U.S.) , Células Madre Embrionarias/fisiología , Humanos , Apoyo a la Investigación como Asunto , Estados UnidosRESUMEN
A recessive deafness mutation in the mouse arose spontaneously and was identified in a colony segregating a null allele of the gastrin-releasing peptide receptor (Grpr) locus. Auditory-evoked brain stem response measurements revealed deafness in 7-week-old affected mice. By linkage analyses, the mutant phenotype was mapped near marker D10Mit186 and the protocadherin gene Pcdh15. As shown by complementation testing, the new mutation is allelic with Ames waltzer (Pcdh15(av)). Sequencing mutant-derived brain Pcdh15 cDNAs identified the insertion of a cytosine residue at nucleotide position c2099 (2099insC), which results in a frame-shift and premature stop codon. Abnormal stereocilia on inner and outer hair cells of the organ of Corti were identified by scanning electron microscopy as early as postnatal day 0 and cross-sectional histology revealed severe neuroepithelial degeneration in cochleas of 30-50-day-old mutants. The new allele of Ames waltzer, designated Pcdh15(av-Jfb), may aid in studying the histopathology associated with Usher syndrome type 1F, which is caused by a functional null allele of PCDH15.
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Cadherinas/genética , Sordera/genética , Mutación del Sistema de Lectura/genética , Trastornos del Movimiento/genética , Precursores de Proteínas/genética , Animales , Umbral Auditivo , Secuencia de Bases/genética , Conducta Animal/fisiología , Proteínas Relacionadas con las Cadherinas , Cóclea/inervación , Cóclea/patología , Citosina , Sordera/patología , Potenciales Evocados Auditivos del Tronco Encefálico , Genes Recesivos , Prueba de Complementación Genética , Ligamiento Genético , Ratones , Ratones Endogámicos , Trastornos del Movimiento/patología , Fenotipo , Ganglio Espiral de la Cóclea/patologíaRESUMEN
Chronic itch, or pruritus, is associated with a wide range of skin abnormalities. The mechanisms responsible for chronic itch induction and persistence remain unclear. We developed a mouse model in which a constitutively active form of the serine/threonine kinase BRAF was expressed in neurons gated by the sodium channel Nav1.8 (BRAF(Nav1.8) mice). We found that constitutive BRAF pathway activation in BRAF(Nav1.8) mice results in ectopic and enhanced expression of a cohort of itch-sensing genes, including gastrin-releasing peptide (GRP) and MAS-related GPCR member A3 (MRGPRA3), in nociceptors expressing transient receptor potential vanilloid 1 (TRPV1). BRAF(Nav1.8) mice showed de novo neuronal responsiveness to pruritogens, enhanced pruriceptor excitability, and heightened evoked and spontaneous scratching behavior. GRP receptor expression was increased in the spinal cord, indicating augmented coding capacity for itch subsequent to amplified pruriceptive inputs. Enhanced GRP expression and sustained ERK phosphorylation were observed in sensory neurons of mice with allergic contact dermatitis or dry skinelicited itch; however, spinal ERK activation was not required for maintaining central sensitization of itch. Inhibition of either BRAF or GRP signaling attenuated itch sensation in chronic itch mouse models. These data uncover RAF/MEK/ERK signaling as a key regulator that confers a subset of nociceptors with pruriceptive properties to initiate and maintain long-lasting itch sensation.
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Proteínas Proto-Oncogénicas B-raf/fisiología , Prurito/etiología , Prurito/fisiopatología , Células Receptoras Sensoriales/fisiología , Animales , Enfermedad Crónica , Modelos Animales de Enfermedad , Péptido Liberador de Gastrina/genética , Péptido Liberador de Gastrina/fisiología , Expresión Génica , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Noqueados , Ratones Transgénicos , Canal de Sodio Activado por Voltaje NAV1.8/genética , Canal de Sodio Activado por Voltaje NAV1.8/fisiología , Nociceptores/fisiología , Proteínas Proto-Oncogénicas B-raf/genética , Prurito/genética , Receptores de Bombesina/genética , Receptores de Bombesina/fisiología , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/fisiología , Médula Espinal/fisiopatología , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/fisiologíaAsunto(s)
Biología/tendencias , Mapeo Encefálico/métodos , Neurociencias/tendencias , Investigación/tendencias , Animales , Redes Comunitarias , Biología Computacional , Redes de Comunicación de Computadores , Conducta Cooperativa , Humanos , Sistemas de Información , Internet , Publicaciones , Proyectos de Investigación , Apoyo SocialAsunto(s)
Otolaringología/educación , Investigación/educación , Selección de Profesión , Curriculum , Docentes Médicos , Humanos , National Institutes of Health (U.S.) , Objetivos Organizacionales , Otolaringología/economía , Otolaringología/organización & administración , Selección de Personal , Investigación/economía , Investigación/organización & administración , Apoyo a la Investigación como Asunto , Sociedades Médicas , Desarrollo de Personal , Estados UnidosRESUMEN
Stem cells have two remarkable properties. They can either renew themselves or they can differentiate into one or more adult cell types. Stem cells derived from a human embryo appear to have an unlimited capacity to self-renew in cell culture, and they are also able to differentiate into hundreds of adult cell types. Human embryonic stem cell lines offer a platform technology that has the potential to elucidate the molecular mechanisms that determine adult cell fate, generate cellular models for discovery of new drugs, and create populations of differentiated cells for novel transplantation therapies. The National Institutes of Health (NIH) has identified some of the rate-limiting steps toward realizing this potential, and has forged funding initiatives to accelerate research progress. Given the remarkable potential, NIH support for research using stem cells is an important priority for the foreseeable future.
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Células Madre Pluripotentes/citología , Células Madre Pluripotentes/fisiología , Diferenciación Celular , Línea Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/fisiología , Humanos , National Institutes of Health (U.S.) , Investigación/legislación & jurisprudencia , Investigación/tendencias , Apoyo a la Investigación como Asunto , Trasplante de Células Madre , Estados UnidosRESUMEN
Specialization in cell function and morphology is influenced by the differential expression of mRNAs, many of which are expressed at low abundance and restricted to certain cell types. Detecting such transcripts in cDNA libraries may require sequencing millions of clones. Massively parallel signature sequencing (MPSS) is well suited to identifying transcripts that are expressed in discrete cell types and in low abundance. We have made MPSS libraries from microdissections of three inner ear tissues. By comparing these MPSS libraries to those of 87 other tissues included in the Mouse Reference Transcriptome online resource, we have identified genes that are highly enriched in, or specific to, the inner ear. We show by RT-PCR and in situ hybridization that signatures unique to the inner ear libraries identify transcripts with highly specific cell-type localizations. These transcripts serve to illustrate the utility of a resource that is available to the research community. Utilization of these resources will increase the number of known transcription units and expand our knowledge of the tissue-specific regulation of the transcriptome.
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Oído Interno/metabolismo , Biblioteca de Genes , ARN Mensajero/genética , ARN Mensajero/metabolismo , Animales , Secuencia de Bases , ADN/genética , Femenino , Expresión Génica , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Distribución TisularRESUMEN
The human T1R taste receptors are family C G-protein-coupled receptors (GPCRs) that act as heterodimers to mediate sweet (hT1R2 + hT1R3) and umami (hT1R1 + hT1R3) taste modalities. Each T1R has a large extracellular ligand-binding domain linked to a seven transmembrane-spanning core domain (7TMD). We demonstrate that the 7TMDs of hT1R1 and hT1R2 display robust ligand-independent constitutive activity, efficiently catalyzing the exchange of GDP for GTP on Galpha subunits. In contrast, relative to the 7TMDs of hT1R1 and hT1R2, the 7TMD of hT1R3 couples poorly to G-proteins, suggesting that in vivo signaling may proceed primarily through hT1R1 and hT1R2. In addition, we provide direct evidence that the hT1Rs selectively signal through Galpha(i/o) pathways, coupling to multiple Galpha(i/o) subunits as well as the taste cell specific Gbeta(1)gamma(13) dimer.