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1.
Mamm Genome ; 34(3): 453-463, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37341808

RESUMO

The external ear develops from an organized convergence of ventrally migrating neural crest cells into the first and second branchial arches. Defects in external ear position are often symptomatic of complex syndromes such as Apert, Treacher-Collins, and Crouzon Syndrome. The low set ears (Lse) spontaneous mouse mutant is characterized by the dominant inheritance of a ventrally shifted external ear position and an abnormal external auditory meatus (EAM). We identified the causative mutation as a 148 Kb tandem duplication on Chromosome 7, which includes the entire coding sequences of Fgf3 and Fgf4. Duplications of FGF3 and FGF4 occur in 11q duplication syndrome in humans and are associated with craniofacial anomalies, among other features. Intercrosses of Lse-affected mice revealed perinatal lethality in homozygotes, and Lse/Lse embryos display additional phenotypes including polydactyly, abnormal eye morphology, and cleft secondary palate. The duplication results in increased Fgf3 and Fgf4 expression in the branchial arches and additional discrete domains in the developing embryo. This ectopic overexpression resulted in functional FGF signaling, demonstrated by increased Spry2 and Etv5 expression in overlapping domains of the developing arches. Finally, a genetic interaction between Fgf3/4 overexpression and Twist1, a regulator of skull suture development, resulted in perinatal lethality, cleft palate, and polydactyly in compound heterozygotes. These data indicate a role for Fgf3 and Fgf4 in external ear and palate development and provide a novel mouse model for further interrogation of the biological consequences of human FGF3/4 duplication.


Assuntos
Fatores de Crescimento de Fibroblastos , Polidactilia , Animais , Camundongos , Humanos , Fatores de Crescimento de Fibroblastos/genética , Mutação , Modelos Animais de Doenças , Fator 3 de Crescimento de Fibroblastos/genética
2.
PLoS Genet ; 12(2): e1005805, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26828925

RESUMO

The extent and strength of epistasis is commonly unresolved in genetic studies, and observed epistasis is often difficult to interpret in terms of biological consequences or overall genetic architecture. We investigated the prevalence and consequences of epistasis by analyzing four body composition phenotypes--body weight, body fat percentage, femoral density, and femoral circumference--in a large F2 intercross of B6-lit/lit and C3.B6-lit/lit mice. We used Combined Analysis of Pleiotropy and Epistasis (CAPE) to examine interactions for the four phenotypes simultaneously, which revealed an extensive directed network of genetic loci interacting with each other, circulating IGF1, and sex to influence these phenotypes. The majority of epistatic interactions had small effects relative to additive effects of individual loci, and tended to stabilize phenotypes towards the mean of the population rather than extremes. Interactive effects of two alleles inherited from one parental strain commonly resulted in phenotypes closer to the population mean than the additive effects from the two loci, and often much closer to the mean than either single-locus model. Alternatively, combinations of alleles inherited from different parent strains contribute to more extreme phenotypes not observed in either parental strain. This class of phenotype-stabilizing interactions has effects that are close to additive and are thus difficult to detect except in very large intercrosses. Nevertheless, we found these interactions to be useful in generating hypotheses for functional relationships between genetic loci. Our findings suggest that while epistasis is often weak and unlikely to account for a large proportion of heritable variance, even small-effect genetic interactions can facilitate hypotheses of underlying biology in well-powered studies.


Assuntos
Cruzamentos Genéticos , Epistasia Genética , Motivos de Aminoácidos , Animais , Composição Corporal , Densidade Óssea , Osso e Ossos/fisiologia , Feminino , Redes Reguladoras de Genes , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Fenótipo , Locos de Características Quantitativas/genética , Fator de Células-Tronco/metabolismo
3.
Genome Res ; 25(7): 948-57, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25917818

RESUMO

Spontaneously arising mouse mutations have served as the foundation for understanding gene function for more than 100 years. We have used exome sequencing in an effort to identify the causative mutations for 172 distinct, spontaneously arising mouse models of Mendelian disorders, including a broad range of clinically relevant phenotypes. To analyze the resulting data, we developed an analytics pipeline that is optimized for mouse exome data and a variation database that allows for reproducible, user-defined data mining as well as nomination of mutation candidates through knowledge-based integration of sample and variant data. Using these new tools, putative pathogenic mutations were identified for 91 (53%) of the strains in our study. Despite the increased power offered by potentially unlimited pedigrees and controlled breeding, about half of our exome cases remained unsolved. Using a combination of manual analyses of exome alignments and whole-genome sequencing, we provide evidence that a large fraction of unsolved exome cases have underlying structural mutations. This result directly informs efforts to investigate the similar proportion of apparently Mendelian human phenotypes that are recalcitrant to exome sequencing.


Assuntos
Exoma , Mutação , Animais , Feminino , Doenças Genéticas Inatas/genética , Ligação Genética , Variação Genética , Estudo de Associação Genômica Ampla , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Masculino , Camundongos , Fenótipo , Reprodutibilidade dos Testes
4.
Dev Biol ; 415(2): 216-227, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-26234751

RESUMO

Craniofacial abnormalities are among the most common features of human genetic syndromes and disorders. The etiology of these conditions is often complex, influenced by both genetic context and the environment. Frequently, craniofacial abnormalities present as part of a syndrome with clear comorbid phenotypes, providing additional insight into mechanisms of the causative gene or pathway. The mouse has been a key tool in our understanding of the genetic mechanisms of craniofacial development and disease, and can provide excellent models for human craniofacial abnormalities. While powerful genetic engineering tools in the mouse have contributed significantly our understanding of craniofacial development and dysmorphology, forward genetic approaches provide an unbiased means to identify new genes and pathways. Moreover, spontaneous mutations can occur on any number of genetic backgrounds, potentially revealing critical genes that require a specific genetic context. Here we report discovery and phenotyping of 43 craniofacial mouse models, derived primarily from a screen for spontaneous mutations in production colonies at the Jackson Laboratory. We identify the causative gene for 33 lines, including novel genes in pathways not previously connected to craniofacial development, and novel alleles of known genes that present with unique phenotypes. Together with our detailed characterization, this work provides a valuable gene discovery resource for the craniofacial community, and a rich source of mouse models for further investigation.


Assuntos
Anormalidades Craniofaciais/genética , Modelos Animais de Doenças , Estudos de Associação Genética , Desenvolvimento Maxilofacial/genética , Camundongos/genética , Alelos , Animais , Cefalometria , Anormalidades Craniofaciais/diagnóstico por imagem , Exoma , Face/anormalidades , Feminino , Redes Reguladoras de Genes , Humanos , Imageamento Tridimensional , Masculino , Mutação , Osteopetrose/genética , Fenótipo , Crânio/anormalidades , Crânio/diagnóstico por imagem , Erupção Dentária/genética , Microtomografia por Raio-X
5.
Nature ; 477(7364): 289-94, 2011 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-21921910

RESUMO

We report genome sequences of 17 inbred strains of laboratory mice and identify almost ten times more variants than previously known. We use these genomes to explore the phylogenetic history of the laboratory mouse and to examine the functional consequences of allele-specific variation on transcript abundance, revealing that at least 12% of transcripts show a significant tissue-specific expression bias. By identifying candidate functional variants at 718 quantitative trait loci we show that the molecular nature of functional variants and their position relative to genes vary according to the effect size of the locus. These sequences provide a starting point for a new era in the functional analysis of a key model organism.


Assuntos
Regulação da Expressão Gênica/genética , Variação Genética/genética , Genoma/genética , Camundongos Endogâmicos/genética , Camundongos/genética , Fenótipo , Alelos , Animais , Animais de Laboratório/genética , Genômica , Camundongos/classificação , Camundongos Endogâmicos C57BL/genética , Filogenia , Locos de Características Quantitativas/genética
6.
Mamm Genome ; 27(1-2): 47-61, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26546009

RESUMO

Genetic variations mediate skeletal responsiveness to mechanical unloading, with individual space travelers exhibiting large variations in the extent of bone loss. We previously identified genomic regions harboring several hundred genes that can modulate the magnitude of skeletal adaptation to mechanical unloading. Here, bioinformatic filters aided in shortlisting 30 genes with bone-related and mechanoregulatory roles. The genes CD44, FGF2, NOD2, and Fas, all associated with ERK signaling, were then functionally tested in hindlimb-unloaded (HLU) knockout (KO) mice. Compared to their respective normally ambulating wildtype (WT) controls, all KO strains, except Fas mice, had lower trabecular bone volume, bone volume fraction, and/or trabecular number. For cortical bone and compared to ambulatory WT mice, CD44(-/-) had impaired properties while FGF2(-/-) showed enhanced indices. NOD2(-/-) and Fas(-/-) did not have a cortical phenotype. In all KO and WT groups, HLU resulted in impaired trabecular and cortical indices, primarily due to trabecular tissue loss and mitigation of cortical bone growth. The difference in trabecular separation between HLU and ambulatory controls was significantly greater in CD44(-/-) and NOD2(-/-) mice than in WT mice. In cortical bone, differences in cortical thickness, total pore volume, and cortical porosity between HLU and controls were aggravated in CD44(-/-) mice. In contrast, deletion of NOD2 and Fas genes mitigated the differences in Po.V between HLU and control mice. Together, we narrowed a previous list of QTL-derived candidate genes from over 300 to 30, and showed that CD44, NOD2, and Fas have distinct functions in regulating changes in trabecular and cortical bone indices during unloading.


Assuntos
Reabsorção Óssea/genética , Fêmur/metabolismo , Receptores de Hialuronatos/genética , Sistema de Sinalização das MAP Quinases , Proteína Adaptadora de Sinalização NOD2/genética , Receptor fas/genética , Animais , Densidade Óssea , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Biologia Computacional , Feminino , Fêmur/patologia , Fator 2 de Crescimento de Fibroblastos/deficiência , Fator 2 de Crescimento de Fibroblastos/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Elevação dos Membros Posteriores , Receptores de Hialuronatos/metabolismo , Masculino , Mecanotransdução Celular , Camundongos , Camundongos Knockout , Proteína Adaptadora de Sinalização NOD2/deficiência , Locos de Características Quantitativas , Microtomografia por Raio-X , Receptor fas/deficiência
7.
BMC Genomics ; 16: 493, 2015 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-26138817

RESUMO

BACKGROUND: Understanding cellular structure and organization, which plays an important role in biological systems ranging from mechanosensation to neural organization, is a complicated multifactorial problem depending on genetics, environmental factors, and stochastic processes. Isolating these factors necessitates the measurement and sensitive quantification of many samples in a reliable, high-throughput, unbiased manner. In this manuscript we present a pipelined approach using a fully automated framework based on Synchrotron-based X-ray Tomographic Microscopy (SRXTM) for performing a full 3D characterization of millions of substructures. RESULTS: We demonstrate the framework on a genetic study on the femur bones of in-bred mice. We measured 1300 femurs from a F2 cross experiment in mice without the growth hormone (which can confound many of the smaller structural differences between strains) and characterized more than 50 million osteocyte lacunae (cell-sized hollows in the bone). The results were then correlated with genetic markers in a process called quantitative trait localization (QTL). Our findings provide a mapping between regions of the genome (all 19 autosomes) and observable phenotypes which could explain between 8-40 % of the variance using between 2-10 loci for each trait. This map shows 4 areas of overlap with previous studies looking at bone strength and 3 areas not previously associated with bone. CONCLUSIONS: The mapping of microstructural phenotypes provides a starting point for both structure-function and genetic studies on murine bone structure and the specific loci can be investigated in more detail to identify single gene candidates which can then be translated to human investigations. The flexible infrastructure offers a full spectrum of shape, distribution, and connectivity metrics for cellular networks and can be adapted to a wide variety of materials ranging from plant roots to lung tissue in studies requiring high sample counts and sensitive metrics such as the drug-gene interactions and high-throughput screening.


Assuntos
Fêmur/anatomia & histologia , Fêmur/diagnóstico por imagem , Ligação Genética , Imageamento Tridimensional/métodos , Tomografia por Raios X/métodos , Animais , Densidade Óssea , Mapeamento Cromossômico , Camundongos , Fenótipo , Locos de Características Quantitativas , Síncrotrons
8.
Mamm Genome ; 26(3-4): 173-80, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25673119

RESUMO

Normal fusion of developing eyelids requires coordination of inductive signals from the eyelid mesenchyme with migration of the periderm cell layer and constriction of the eyelids across the eye. Failure of this process results in an eyelids open at birth (EOB) phenotype in mice. We have identified a novel spontaneous allele of Alx4 that displays EOB, in addition to polydactyly and cranial malformations. Alx4 is expressed in the eyelid mesenchyme prior to and during eyelid fusion in a domain overlapping the expression of genes that also play a role in normal eyelid development. We show that Alx4 mutant mice have reduced expression of Fgf10, a key factor expressed in the mesenchyme that is required for initiation of eyelid fusion by the periderm. This is accompanied by a reduced number of periderm cells expressing phosphorylated c-Jun, consistent with the incomplete ablation of Fgf10 expression. Together, these data demonstrate that eyelid fusion in mice requires the expression of Alx4, accompanied by the loss of normal expression of essential components of the eyelid fusion pathway.


Assuntos
Alelos , Epistasia Genética , Pálpebras/patologia , Fator 10 de Crescimento de Fibroblastos/genética , Proteínas de Homeodomínio/genética , Fenótipo , Animais , Sequência de Bases , Pálpebras/embriologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Ordem dos Genes , Marcação de Genes , Estudos de Associação Genética , Masculino , Mesoderma/embriologia , Mesoderma/metabolismo , Camundongos , Locos de Características Quantitativas , Deleção de Sequência
9.
Nat Genet ; 38(2): 245-50, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16380713

RESUMO

Hypertonia, which results from motor pathway defects in the central nervous system (CNS), is observed in numerous neurological conditions, including cerebral palsy, stroke, spinal cord injury, stiff-person syndrome, spastic paraplegia, dystonia and Parkinson disease. Mice with mutation in the hypertonic (hyrt) gene exhibit severe hypertonia as their primary symptom. Here we show that hyrt mutant mice have much lower levels of gamma-aminobutyric acid type A (GABA(A)) receptors in their CNS, particularly the lower motor neurons, than do wild-type mice, indicating that the hypertonicity of the mutants is likely to be caused by deficits in GABA-mediated motor neuron inhibition. We cloned the responsible gene, trafficking protein, kinesin binding 1 (Trak1), and showed that its protein product interacts with GABA(A) receptors. Our data implicate Trak1 as a crucial regulator of GABA(A) receptor homeostasis and underscore the importance of hyrt mice as a model for studying the molecular etiology of hypertonia associated with human neurological diseases.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Homeostase , Hipertonia Muscular/metabolismo , Mutação/genética , Receptores de GABA-A/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Animais , Células do Corno Anterior/patologia , Cromossomos de Mamíferos/genética , Diazepam/farmacologia , Eletromiografia , Expressão Gênica , Homozigoto , Humanos , Corpos de Inclusão/patologia , Corpos de Inclusão/ultraestrutura , Camundongos , Dados de Sequência Molecular , Hipertonia Muscular/genética , Hipertonia Muscular/patologia , Músculo Esquelético/efeitos dos fármacos , Mapeamento Físico do Cromossomo , Ponte/patologia , Ponte/ultraestrutura , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
10.
Proc Natl Acad Sci U S A ; 108(44): 18108-13, 2011 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-22025706

RESUMO

The anorectic anx/anx mouse exhibits disturbed feeding behavior and aberrances, including neurodegeneration, in peptidergic neurons in the appetite regulating hypothalamic arcuate nucleus. Poor feeding in infants, as well as neurodegeneration, are common phenotypes in human disorders caused by dysfunction of the mitochondrial oxidative phosphorylation system (OXPHOS). We therefore hypothesized that the anorexia and degenerative phenotypes in the anx/anx mouse could be related to defects in the OXPHOS. In this study, we found reduced efficiency of hypothalamic OXPHOS complex I assembly and activity in the anx/anx mouse. We also recorded signs of increased oxidative stress in anx/anx hypothalamus, possibly as an effect of the decreased hypothalamic levels of fully assembled complex I, that were demonstrated by native Western blots. Furthermore, the Ndufaf1 gene, encoding a complex I assembly factor, was genetically mapped to the anx interval and found to be down-regulated in anx/anx mice. These results suggest that the anorexia and hypothalamic neurodegeneration of the anx/anx mouse are associated with dysfunction of mitochondrial complex I.


Assuntos
Anorexia/fisiopatologia , Hipotálamo/fisiopatologia , Mitocôndrias/fisiologia , Alelos , Animais , Anorexia/genética , Hipotálamo/metabolismo , Camundongos , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Estresse Oxidativo
11.
Dev Biol ; 355(2): 175-82, 2011 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-21458441

RESUMO

The FaceBase Consortium consists of ten interlinked research and technology projects whose goal is to generate craniofacial research data and technology for use by the research community through a central data management and integrated bioinformatics hub. Funded by the National Institute of Dental and Craniofacial Research (NIDCR) and currently focused on studying the development of the middle region of the face, the Consortium will produce comprehensive datasets of global gene expression patterns, regulatory elements and sequencing; will generate anatomical and molecular atlases; will provide human normative facial data and other phenotypes; conduct follow up studies of a completed genome-wide association study; generate independent data on the genetics of craniofacial development, build repositories of animal models and of human samples and data for community access and analysis; and will develop software tools and animal models for analyzing and functionally testing and integrating these data. The FaceBase website (http://www.facebase.org) will serve as a web home for these efforts, providing interactive tools for exploring these datasets, together with discussion forums and other services to support and foster collaboration within the craniofacial research community.


Assuntos
Biologia Computacional/métodos , Bases de Dados Factuais , Face/embriologia , Perfilação da Expressão Gênica , Pesquisa , Crânio/embriologia , Humanos , Software
12.
Mamm Genome ; 23(9-10): 559-71, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22945696

RESUMO

Because the mouse is used so widely for biomedical research and the number of mouse models being generated is increasing rapidly, centralized repositories are essential if the valuable mouse strains and models that have been developed are to be securely preserved and fully exploited. Ensuring the ongoing availability of these mouse strains preserves the investment made in creating and characterizing them and creates a global resource of enormous value. The establishment of centralized mouse repositories around the world for distributing and archiving these resources has provided critical access to and preservation of these strains. This article describes the common and specialized activities provided by major mouse repositories around the world.


Assuntos
Camundongos/genética , Animais , Controle de Qualidade , Especificidade da Espécie
13.
Blood ; 115(6): 1267-76, 2010 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-19846887

RESUMO

The spontaneous mouse mutation "thrombocytopenia and cardiomyopathy" (trac) causes macrothrombocytopenia, prolonged bleeding times, anemia, leukopenia, infertility, cardiomyopathy, and shortened life span. Homozygotes show a 20-fold decrease in platelet numbers and a 3-fold increase in platelet size with structural alterations and functional impairments in activation and aggregation. Megakaryocytes in trac/trac mice are present in increased numbers, have poorly developed demarcation membrane systems, and have decreased polyploidy. The thrombocytopenia is not intrinsic to defects at the level of hematopoietic progenitor cells but is associated with a microenvironmental abnormality. The trac mutation maps to mouse chromosome 17, syntenic with human chromosome 2p21-22. A G to A mutation in exon 10 of the adenosine triphosphate (ATP)-binding cassette subfamily G, member 5 (Abcg5) gene, alters a tryptophan codon (UGG) to a premature stop codon (UAG). Crosses with mice doubly transgenic for the human ABCG5 and ABCG8 genes rescued platelet counts and volumes. ABCG5 and ABCG8 form a functional complex that limits dietary phytosterol accumulation. Phytosterolemia in trac/trac mice confirmed a functional defect in the ABCG5/ABCG8 transport system. The trac mutation provides a new clinically significant animal model for human phytosterolemia and provides a new means for studying the role of phytosterols in hematologic diseases and testing therapeutic interventions.


Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Cardiomiopatias/genética , Modelos Animais de Doenças , Erros Inatos do Metabolismo Lipídico/genética , Lipoproteínas/fisiologia , Mutação/genética , Fitosteróis/metabolismo , Sitosteroides/metabolismo , Trombocitopenia/genética , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Tempo de Sangramento , Cardiomiopatias/patologia , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Cruzamentos Genéticos , Feminino , Feto/citologia , Feto/metabolismo , Erros Inatos do Metabolismo Lipídico/patologia , Lipoproteínas/genética , Fígado/citologia , Fígado/metabolismo , Masculino , Megacariócitos/citologia , Megacariócitos/metabolismo , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Trombocitopenia/patologia
14.
Mamm Genome ; 22(11-12): 685-91, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21953412

RESUMO

Ts65Dn is a mouse model of Down syndrome: a syndrome that results from chromosome (Chr) 21 trisomy and is associated with congenital defects, cognitive impairment, and ultimately Alzheimer's disease. Ts65Dn mice have segmental trisomy for distal mouse Chr 16, a region sharing conserved synteny with human Chr 21. As a result, this strain harbors three copies of over half of the human Chr 21 orthologs. The trisomic segment of Chr 16 is present as a translocation chromosome (Mmu17(16)), with breakpoints that have not been defined previously. To molecularly characterize the Chrs 16 and 17 breakpoints on the translocation chromosome in Ts65Dn mice, we used a selective enrichment and high-throughput paired-end sequencing approach. Analysis of paired-end reads flanking the Chr 16, Chr 17 junction on Mmu17(16) and de novo assembly of the reads directly spanning the junction provided the precise locations of the Chrs 16 and 17 breakpoints at 84,351,351 and 9,426,822 bp, respectively. These data provide the basis for low-cost, highly efficient genotyping of Ts65Dn mice. More importantly, these data provide, for the first time, complete characterization of gene dosage in Ts65Dn mice.


Assuntos
Modelos Animais de Doenças , Síndrome de Down/genética , Translocação Genética , Trissomia , Animais , Sequência de Bases , Síndrome de Down/patologia , Feminino , Dosagem de Genes , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Polimorfismo de Nucleotídeo Único , Alinhamento de Sequência , Análise de Sequência de DNA
15.
Differentiation ; 80(1): 9-19, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20537458

RESUMO

While endogenous Myc (c-myc) and Mycn (N-myc) have been reported to be separately dispensable for murine embryonic stem cell (mESC) function, myc greatly enhances induced pluripotent stem (iPS) cell formation and overexpressed c-myc confers LIF-independence upon mESC. To address the role of myc genes in ESC and in pluripotency generally, we conditionally knocked out both c- and N-myc using myc doubly homozygously floxed mESC lines (cDKO). Both lines of myc cDKO mESC exhibited severely disrupted self-renewal, pluripotency, and survival along with enhanced differentiation. Chimeric embryos injected with DKO mESC most often completely failed to develop or in rare cases survived but with severe defects. The essential nature of myc for self-renewal and pluripotency is at least in part mediated through orchestrating pluripotency-related cell cycle and metabolic programs. This study demonstrates that endogenous myc genes are essential for mESC pluripotency and self-renewal as well as providing the first evidence that myc genes are required for early embryogenesis, suggesting potential mechanisms of myc contribution to iPS cell formation.


Assuntos
Diferenciação Celular , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Genes myc/fisiologia , Células-Tronco Pluripotentes/citologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Animais , Biomarcadores/metabolismo , Western Blotting , Ciclo Celular , Linhagem da Célula , Proliferação de Células , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Células-Tronco Embrionárias/metabolismo , Feminino , Citometria de Fluxo , Imunofluorescência , Perfilação da Expressão Gênica , Técnicas Imunoenzimáticas , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/metabolismo , Masculino , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Células-Tronco Pluripotentes/metabolismo , RNA Mensageiro/genética , Regeneração , Reação em Cadeia da Polimerase Via Transcriptase Reversa
16.
Mamm Genome ; 20(7): 424-36, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19629596

RESUMO

Forward genetics (phenotype-driven approaches) remain the primary source for allelic variants in the mouse. Unfortunately, the gap between observable phenotype and causative genotype limits the widespread use of spontaneous and induced mouse mutants. As alternatives to traditional positional cloning and mutation detection approaches, sequence capture and next-generation sequencing technologies can be used to rapidly sequence subsets of the genome. Application of these technologies to mutation detection efforts in the mouse has the potential to significantly reduce the time and resources required for mutation identification by abrogating the need for high-resolution genetic mapping, long-range PCR, and sequencing of individual PCR amplimers. As proof of principle, we used array-based sequence capture and pyrosequencing to sequence an allelic series from the classically defined Kit locus (approximately 200 kb) from each of five noncomplementing Kit mutants (one known allele and four unknown alleles) and have successfully identified and validated a nonsynonymous coding mutation for each allele. These data represent the first documentation and validation that these new technologies can be used to efficiently discover causative mutations. Importantly, these data also provide a specific methodological foundation for the development of large-scale mutation detection efforts in the laboratory mouse.


Assuntos
Análise Mutacional de DNA/métodos , Camundongos/genética , Mutação , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Dados de Sequência Molecular , Alinhamento de Sequência
17.
Mamm Genome ; 20(8): 462-75, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19669234

RESUMO

Podosome-type adhesions are actin-based membrane protrusions involved in cell-matrix adhesion and extracellular matrix degradation. Despite growing knowledge of many proteins associated with podosome-type adhesions, much remains unknown concerning the function of podosomal proteins at the level of the whole animal. In this study, the spontaneous mouse mutant nee was used to identify a component of podosome-type adhesions that is essential for normal postnatal growth and development. Mice homozygous for the nee allele exhibited runted growth, craniofacial and skeletal abnormalities, ocular anterior segment dysgenesis, and hearing impairment. Adults also exhibited infertility and a form of lipodystrophy. Using genetic mapping and DNA sequencing, the cause of nee phenotypes was identified as a 1-bp deletion within the Sh3pxd2b gene on mouse Chromosome 11. Whereas the wild-type Sh3pxd2b gene is predicted to encode a protein with one PX domain and four SH3 domains, the nee mutation is predicted to cause a frameshift and a protein truncation altering a portion of the third SH3 domain and deleting all of the fourth SH3 domain. The SH3PXD2B protein is believed to be an important component of podosomes likely to mediate protein-protein interactions with membrane-spanning metalloproteinases. Testing this directly, SH3PXD2B localized to podosomes in constitutively active Src-transfected fibroblasts and through its last SH3 domain associated with a transmembrane member of a disintegrin and metalloproteinase family of proteins, ADAM15. These results identify SH3PXD2B as a podosomal-adaptor protein required for postnatal growth and development, particularly within physiologic contexts involving extracellular matrix regulation.


Assuntos
Extensões da Superfície Celular/metabolismo , Camundongos/crescimento & desenvolvimento , Proteínas de Transferência de Fosfolipídeos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Extensões da Superfície Celular/química , Extensões da Superfície Celular/genética , Mapeamento Cromossômico , Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/metabolismo , Anormalidades do Olho/genética , Anormalidades do Olho/metabolismo , Feminino , Masculino , Camundongos/genética , Camundongos/metabolismo , Dados de Sequência Molecular , Mutação , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/genética , Transporte Proteico , Alinhamento de Sequência
18.
Physiol Genomics ; 33(1): 26-32, 2008 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-18230669

RESUMO

Laboratory inbred mouse strains show a broad range of variation in phenotypes, such as body composition, bone mineral density (BMD), plasma leptin, and insulin-like growth factor I (IGF-I), and thus provide a basis for the study of associations among them. We analyzed these phenotypes in male and female mice from 43 inbred strains fed on a high-fat (30% caloric content) diet and from 30 inbred strains fed on a low-fat (6%) diet. Structural equation modeling of these data reveals that the relationship of body fat content and areal BMD is altered by dietary factors and genotypes. Sex has no net effect on areal BMD, but after accounting for body mass difference females have higher areal BMD. Leptin is affected by relative fat mass and has no net effect on areal BMD. IGF-I has a direct effect on areal BMD.


Assuntos
Composição Corporal/efeitos dos fármacos , Densidade Óssea/efeitos dos fármacos , Gorduras na Dieta/farmacologia , Camundongos Endogâmicos/fisiologia , Animais , Dieta Aterogênica , Feminino , Fator de Crescimento Insulin-Like I/fisiologia , Leptina/sangue , Leptina/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos/sangue , Modelos Biológicos , Fatores Sexuais
19.
Mol Endocrinol ; 21(7): 1593-602, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17440044

RESUMO

Dual oxidases generate the hydrogen peroxide needed by thyroid peroxidase for the incorporation of iodine into thyroglobulin, an essential step in thyroid hormone synthesis. Mutations in the human dual oxidase 2 gene, DUOX2, have been shown to underlie several cases of congenital hypothyroidism. We report here the first mouse Duox2 mutation, which provides a new genetic model for studying the specific function of DUOX2 in the thyroid gland and in other organ systems where it is hypothesized to play a role. We mapped the new spontaneous mouse mutation to chromosome 2 and identified it as a T>G base pair change in exon 16 of Duox2. The mutation changes a highly conserved valine to glycine at amino acid position 674 (V674G) and was named "thyroid dyshormonogenesis" (symbol thyd) to signify a defect in thyroid hormone synthesis. Thyroid glands of mutant mice are goitrous and contain few normal follicles, and anterior pituitaries are dysplastic. Serum T(4) in homozygotes is about one-tenth the level of controls and is accompanied by a more than 100-fold increase in TSH. The weight of adult mutant mice is approximately half that of littermate controls, and serum IGF-I is reduced. The cochleae of mutant mice exhibit abnormalities characteristic of hypothyroidism, including a delayed formation of the inner sulcus and tunnel of Corti and an abnormally thickened tectorial membrane. Hearing thresholds of adult mutant mice are on average 50-60 decibels (dB) above those of controls.


Assuntos
Hipotireoidismo Congênito/enzimologia , Hipotireoidismo Congênito/genética , Nanismo/enzimologia , Nanismo/genética , Flavoproteínas/genética , Perda Auditiva/enzimologia , Perda Auditiva/genética , Mutação de Sentido Incorreto , NADPH Oxidases/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sequência de Bases , Peso Corporal , Cóclea/patologia , Hipotireoidismo Congênito/sangue , Hipotireoidismo Congênito/patologia , Primers do DNA/genética , Modelos Animais de Doenças , Oxidases Duais , Feminino , Flavoproteínas/química , Flavoproteínas/fisiologia , Perda Auditiva/patologia , Perda Auditiva/fisiopatologia , Homozigoto , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Dados de Sequência Molecular , NADPH Oxidases/química , NADPH Oxidases/fisiologia , Fenótipo , Gravidez , Homologia de Sequência de Aminoácidos , Tireotropina/sangue , Tiroxina/sangue
20.
Evodevo ; 9: 3, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29423138

RESUMO

BACKGROUND: Previous analysis suggested that the relative contribution of individual bones to regional skull lengths differ between inbred mouse strains. If the negative correlation of adjacent bone lengths is associated with genetic variation in a heterogeneous population, it would be an example of negative pleiotropy, which occurs when a genetic factor leads to opposite effects in two phenotypes. Confirming negative pleiotropy and determining its basis may reveal important information about the maintenance of overall skull integration and developmental constraint on skull morphology. RESULTS: We identified negative correlations between the lengths of the frontal and parietal bones in the midline cranial vault as well as the zygomatic bone and zygomatic process of the maxilla, which contribute to the zygomatic arch. Through gene association mapping of a large heterogeneous population of Diversity Outbred (DO) mice, we identified a quantitative trait locus on chromosome 17 driving the antagonistic contribution of these two zygomatic arch bones to total zygomatic arch length. Candidate genes in this region were identified and real-time PCR of the maxillary processes of DO founder strain embryos indicated differences in the RNA expression levels for two of the candidate genes, Camkmt and Six2. CONCLUSIONS: A genomic region underlying negative pleiotropy of two zygomatic arch bones was identified, which provides a mechanism for antagonism in component bone lengths while constraining overall zygomatic arch length. This type of mechanism may have led to variation in the contribution of individual bones to the zygomatic arch noted across mammals. Given that similar genetic and developmental mechanisms may underlie negative correlations in other parts of the skull, these results provide an important step toward understanding the developmental basis of evolutionary variation and constraint in skull morphology.

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