Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 24
Filtrar
Más filtros










Base de datos
Intervalo de año de publicación
1.
Genetics ; 2021 Nov 22.
Artículo en Inglés | MEDLINE | ID: mdl-34850872

RESUMEN

Primary cilia are sensory and signaling hubs with a protein composition that is distinct from the rest of the cell due to the barrier function of the transition zone (TZ) at the base of the cilium. Protein transport across the TZ is mediated in part by the BBSome, and mutations disrupting TZ and BBSome proteins cause human ciliopathy syndromes. Ciliopathies have phenotypic variability even among patients with identical genetic variants, suggesting a role for modifier loci. To identify potential ciliopathy modifiers, we performed a mutagenesis screen on nphp-4 mutant Caenorhabditis elegans and uncovered a novel allele of bbs-5. Nphp-4;bbs-5 double mutant worms have phenotypes not observed in either individual mutant strain. To test whether this genetic interaction is conserved, we also analyzed zebrafish and mouse mutants. While Nphp4 mutant zebrafish appeared overtly normal, Bbs5 mutants exhibited scoliosis. When combined, Nphp4;Bbs5 double mutant zebrafish did not exhibit synergistic effects, but the lack of a phenotype in Nphp4 mutants makes interpreting these data difficult. In contrast, Nphp4;Bbs5 double mutant mice were not viable and there were fewer mice than expected carrying three mutant alleles. In addition, postnatal loss of Bbs5 in mice using a conditional allele compromised survival when combined with an Nphp4 allele. As cilia are still formed in the double mutant mice, the exacerbated phenotype is likely a consequence of disrupted ciliary signaling. Collectively, these data support an evolutionarily conserved genetic interaction between Bbs5 and Nphp4 alleles that may contribute to the variability in ciliopathy phenotypes.

2.
Cell Death Dis ; 12(7): 659, 2021 06 30.
Artículo en Inglés | MEDLINE | ID: mdl-34193827

RESUMEN

Cellular stress can lead to several human disease pathologies due to aberrant cell death. The p53 family (tp53, tp63, and tp73) and downstream transcriptional apoptotic target genes (PUMA/BBC3 and NOXA/PMAIP1) have been implicated as mediators of stress signals. To evaluate the importance of key stress response components in vivo, we have generated zebrafish null alleles in puma, noxa, p53, p63, and p73. Utilizing these genetic mutants, we have deciphered that the apoptotic response to genotoxic stress requires p53 and puma, but not p63, p73, or noxa. We also identified a delayed secondary wave of genotoxic stress-induced apoptosis that is p53/puma independent. Contrary to genotoxic stress, ER stress-induced apoptosis requires p63 and puma, but not p53, p73, or noxa. Lastly, the oxidative stress-induced apoptotic response requires p63, and both noxa and puma. Our data also indicate that while the neural tube is poised for apoptosis due to genotoxic stress, the epidermis is poised for apoptosis due to ER and oxidative stress. These data indicate there are convergent as well as unique molecular pathways involved in the different stress responses. The commonality of puma in these stress pathways, and the lack of gross or tumorigenic phenotypes with puma loss suggest that a inhibitor of Puma may have therapeutic application. In addition, we have also generated a knockout of the negative regulator of p53, mdm2 to further evaluate the p53-induced apoptosis. Our data indicate that the p53 null allele completely rescues the mdm2 null lethality, while the puma null completely rescues the mdm2 null apoptosis but only partially rescues the phenotype. Indicating Puma is the key mediator of p53-dependent apoptosis. Interestingly the p53 homozygous null zebrafish develop tumors faster than the previously described p53 homozygous missense mutant zebrafish, suggesting the missense allele may be hypomorphic allele.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Daño del ADN , Estrés del Retículo Endoplásmico , Estrés Oxidativo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Proteínas Reguladoras de la Apoptosis/genética , Estrés del Retículo Endoplásmico/efectos de los fármacos , Regulación de la Expresión Génica , Macrólidos/farmacología , Estrés Oxidativo/efectos de los fármacos , Fosfoproteínas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Transducción de Señal , Acetato de Tetradecanoilforbol/farmacología , Tapsigargina/farmacología , Factores de Tiempo , Transactivadores/genética , Transcripción Genética , Proteína p53 Supresora de Tumor/genética , Rayos X , Pez Cebra/genética , Proteínas de Pez Cebra/genética
3.
Genome Med ; 13(1): 90, 2021 05 21.
Artículo en Inglés | MEDLINE | ID: mdl-34020708

RESUMEN

BACKGROUND: We aimed to define the clinical and variant spectrum and to provide novel molecular insights into the DHX30-associated neurodevelopmental disorder. METHODS: Clinical and genetic data from affected individuals were collected through Facebook-based family support group, GeneMatcher, and our network of collaborators. We investigated the impact of novel missense variants with respect to ATPase and helicase activity, stress granule (SG) formation, global translation, and their effect on embryonic development in zebrafish. SG formation was additionally analyzed in CRISPR/Cas9-mediated DHX30-deficient HEK293T and zebrafish models, along with in vivo behavioral assays. RESULTS: We identified 25 previously unreported individuals, ten of whom carry novel variants, two of which are recurrent, and provide evidence of gonadal mosaicism in one family. All 19 individuals harboring heterozygous missense variants within helicase core motifs (HCMs) have global developmental delay, intellectual disability, severe speech impairment, and gait abnormalities. These variants impair the ATPase and helicase activity of DHX30, trigger SG formation, interfere with global translation, and cause developmental defects in a zebrafish model. Notably, 4 individuals harboring heterozygous variants resulting either in haploinsufficiency or truncated proteins presented with a milder clinical course, similar to an individual harboring a de novo mosaic HCM missense variant. Functionally, we established DHX30 as an ATP-dependent RNA helicase and as an evolutionary conserved factor in SG assembly. Based on the clinical course, the variant location, and type we establish two distinct clinical subtypes. DHX30 loss-of-function variants cause a milder phenotype whereas a severe phenotype is caused by HCM missense variants that, in addition to the loss of ATPase and helicase activity, lead to a detrimental gain-of-function with respect to SG formation. Behavioral characterization of dhx30-deficient zebrafish revealed altered sleep-wake activity and social interaction, partially resembling the human phenotype. CONCLUSIONS: Our study highlights the usefulness of social media to define novel Mendelian disorders and exemplifies how functional analyses accompanied by clinical and genetic findings can define clinically distinct subtypes for ultra-rare disorders. Such approaches require close interdisciplinary collaboration between families/legal representatives of the affected individuals, clinicians, molecular genetics diagnostic laboratories, and research laboratories.

4.
Sci Rep ; 10(1): 19710, 2020 11 12.
Artículo en Inglés | MEDLINE | ID: mdl-33184372

RESUMEN

The four transcription factors OCT4, SOX2, KLF4, and MYC (OSKM) together can convert human fibroblasts to induced pluripotent stem cells (iPSCs). It is, however, perplexing that they can do so only for a rare population of the starting cells with a long latency. Transcription factors (TFs) define identities of both the starting fibroblasts and the end product, iPSCs, and are also of paramount importance for the reprogramming process. It is critical to upregulate or activate the iPSC-enriched TFs while downregulate or silence the fibroblast-enriched TFs. This report explores the initial TF responses to OSKM as the molecular underpinnings for both the potency aspects and the limitation sides of the OSKM reprogramming. The authors first defined the TF reprogramome, i.e., the full complement of TFs to be reprogrammed. Most TFs were resistant to OSKM reprogramming at the initial stages, an observation consistent with the inefficiency and long latency of iPSC reprogramming. Surprisingly, the current analyses also revealed that most of the TFs (at least 83 genes) that did respond to OSKM induction underwent legitimate reprogramming. The initial legitimate transcriptional responses of TFs to OSKM reprogramming were also observed in the reprogramming fibroblasts from a different individual. Such early biased legitimate reprogramming of the responsive TFs aligns well with the robustness aspect of the otherwise inefficient and stochastic OSKM reprogramming.


Asunto(s)
Técnicas de Reprogramación Celular/métodos , Fibroblastos/citología , Células Madre Pluripotentes Inducidas/citología , Factores de Transcripción/genética , Células Cultivadas , Reprogramación Celular/efectos de los fármacos , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Lentivirus/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Proteínas Proto-Oncogénicas c-myc/genética , Factores de Transcripción SOXB1/genética , Factores de Transcripción/metabolismo , Transducción Genética
5.
Am J Physiol Renal Physiol ; 316(3): F414-F425, 2019 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-30566001

RESUMEN

Deficiency in polycystin 1 triggers specific changes in energy metabolism. To determine whether defects in other human cystoproteins have similar effects, we studied extracellular acidification and glucose metabolism in human embryonic kidney (HEK-293) cell lines with polycystic kidney and hepatic disease 1 ( PKHD1) and polycystic kidney disease (PKD) 2 ( PKD2) truncating defects along multiple sites of truncating mutations found in patients with autosomal recessive and dominant PKDs. While neither the PKHD1 or PKD2 gene mutations nor their position enhanced cell proliferation rate in our cell line models, truncating mutations in these genes progressively increased overall extracellular acidification over time ( P < 0.001 for PKHD1 and PKD2 mutations). PKHD1 mutations increased nonglycolytic acidification rate (1.19 vs. 1.03, P = 0.002), consistent with an increase in tricarboxylic acid cycle activity or breakdown of intracellular glycogen. In addition, they increased basal and ATP-linked oxygen consumption rates [7.59 vs. 5.42 ( P = 0.015) and 4.55 vs. 2.98 ( P = 0.004)]. The PKHD1 and PKD2 mutations also altered mitochondrial morphology, resembling the effects of polycystin 1 deficiency. Together, these data suggest that defects in major PKD genes trigger changes in mitochondrial energy metabolism. After validation in in vivo models, these initial observations would indicate potential benefits of targeting energy metabolism in the treatment of PKDs.


Asunto(s)
Metabolismo Energético/genética , Glucosa/metabolismo , Proteínas Quinasas/genética , Receptores de Superficie Celular/genética , Proliferación Celular/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica , Células HEK293 , Humanos , Mutación , Proteínas Quinasas/metabolismo , Receptores de Superficie Celular/metabolismo
6.
J Vis Exp ; (113)2016 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-27501381

RESUMEN

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin condensation, microtubule-kinetochore attachment, chromosome segregation, and cytokinesis in a small time frame. Errors in the delicate process can result in human disease, including birth defects and cancer. Traditional approaches investigating human mitotic disease states often rely on cell culture systems, which lack the natural physiology and developmental/tissue-specific context advantageous when studying human disease. This protocol overcomes many obstacles by providing a way to visualize, with high resolution, chromosome dynamics in a vertebrate system, the zebrafish. This protocol will detail an approach that can be used to obtain dynamic images of dividing cells, which include: in vitro transcription, zebrafish breeding/collecting, embryo embedding, and time-lapse imaging. Optimization and modifications of this protocol are also explored. Using H2A.F/Z-EGFP (labels chromatin) and mCherry-CAAX (labels cell membrane) mRNA-injected embryos, mitosis in AB wild-type, auroraB(hi1045) (,) and esco2(hi2865) mutant zebrafish is visualized. High resolution live imaging in zebrafish allows one to observe multiple mitoses to statistically quantify mitotic defects and timing of mitotic progression. In addition, observation of qualitative aspects that define improper mitotic processes (i.e., congression defects, missegregation of chromosomes, etc.) and improper chromosomal outcomes (i.e., aneuploidy, polyploidy, micronuclei, etc.) are observed. This assay can be applied to the observation of tissue differentiation/development and is amenable to the use of mutant zebrafish and pharmacological agents. Visualization of how defects in mitosis lead to cancer and developmental disorders will greatly enhance understanding of the pathogenesis of disease.


Asunto(s)
Embrión no Mamífero/citología , Mitosis , Imagen de Lapso de Tiempo , Pez Cebra/embriología , Animales , Proteínas de Ciclo Celular , Segregación Cromosómica , Citocinesis , Humanos , Cinetocoros , Microtúbulos
7.
PLoS Genet ; 12(7): e1006220, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-27472056

RESUMEN

Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or 'primary' cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh) signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400). While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8). GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC) protein 4 (DRC4) where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR) to generate one of these human missense variants in mice demonstrated that this allele is likely pathogenic.


Asunto(s)
Tipificación del Cuerpo/genética , Cilios/genética , Síndrome de Kartagener/genética , Proteínas/genética , Animales , Movimiento Celular/genética , Chlamydomonas/genética , Cilios/patología , Proteínas del Citoesqueleto , Citoesqueleto/genética , Modelos Animales de Enfermedad , Extremidades/crecimiento & desarrollo , Extremidades/patología , Predisposición Genética a la Enfermedad , Humanos , Síndrome de Kartagener/patología , Ratones , Microtúbulos/genética , Mutación , Tubo Neural/crecimiento & desarrollo , Tubo Neural/patología , Transducción de Señal/genética
8.
Adv Exp Med Biol ; 916: 61-86, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27165349

RESUMEN

Animal models of tumor initiation and tumor progression are essential components toward understanding cancer and designing/validating future therapies. Zebrafish is a powerful model for studying tumorigenesis and has been successfully exploited in drug discovery. According to the zebrafish reference genome, 82 % of disease-associated genes in the Online Mendelian Inheritance in Man (OMIM) database have clear zebrafish orthologues. Using a variety of large-scale random mutagenesis methods developed to date, zebrafish can provide a unique opportunity to identify gene mutations that may be associated with cancer predisposition. On the other hand, newer technologies enabling targeted mutagenesis can facilitate reverse cancer genetic studies and open the door for complex genetic analysis of tumorigenesis. In this chapter, we will describe the various technologies for conducting genome editing in zebrafish with special emphasis on the approaches to inactivate genes.


Asunto(s)
Modelos Animales de Enfermedad , Neoplasias/genética , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Mutagénesis , Retroviridae/genética , Transgenes , Pez Cebra
9.
Dis Model Mech ; 8(8): 941-55, 2015 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-26044958

RESUMEN

Mutations in ESCO2, one of two establishment of cohesion factors necessary for proper sister chromatid cohesion (SCC), cause a spectrum of developmental defects in the autosomal-recessive disorder Roberts syndrome (RBS), warranting in vivo analysis of the consequence of cohesion dysfunction. Through a genetic screen in zebrafish targeting embryonic-lethal mutants that have increased genomic instability, we have identified an esco2 mutant zebrafish. Utilizing the natural transparency of zebrafish embryos, we have developed a novel technique to observe chromosome dynamics within a single cell during mitosis in a live vertebrate embryo. Within esco2 mutant embryos, we observed premature chromatid separation, a unique chromosome scattering, prolonged mitotic delay, and genomic instability in the form of anaphase bridges and micronuclei formation. Cytogenetic studies indicated complete chromatid separation and high levels of aneuploidy within mutant embryos. Amongst aneuploid spreads, we predominantly observed decreases in chromosome number, suggesting that either cells with micronuclei or micronuclei themselves are eliminated. We also demonstrated that the genomic instability leads to p53-dependent neural tube apoptosis. Surprisingly, although many cells required Esco2 to establish cohesion, 10-20% of cells had only weakened cohesion in the absence of Esco2, suggesting that compensatory cohesion mechanisms exist in these cells that undergo a normal mitotic division. These studies provide a unique in vivo vertebrate view of the mitotic defects and consequences of cohesion establishment loss, and they provide a compensation-based model to explain the RBS phenotypes.


Asunto(s)
Acetiltransferasas/genética , Cromátides/metabolismo , Anomalías Craneofaciales/genética , Anomalías Craneofaciales/patología , Ectromelia/genética , Ectromelia/patología , Hipertelorismo/genética , Hipertelorismo/patología , Mutación/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Acetiltransferasas/deficiencia , Acetiltransferasas/metabolismo , Animales , Apoptosis , Segregación Cromosómica , Cromosomas/metabolismo , Pérdida del Embrión/metabolismo , Pérdida del Embrión/patología , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Inestabilidad Genómica , Índice Mitótico , Modelos Biológicos , Mutagénesis Insercional/genética , Tubo Neural/metabolismo , Tubo Neural/patología , Fenotipo , Retroviridae/genética , Proteína p53 Supresora de Tumor/metabolismo , Pez Cebra/embriología , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/metabolismo
10.
Drug Metab Dispos ; 43(7): 1037-44, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-25934576

RESUMEN

Since its identification in 2000, sulfotransferase (SULT) 4A1 has presented an enigma to the field of cytosolic SULT biology. SULT4A1 is exclusively expressed in neural tissue, is highly conserved, and has been identified in every vertebrate studied to date. Despite this singular level of conservation, no substrate or function for SULT4A1 has been identified. Previous studies demonstrated that SULT4A1 does not bind the obligate sulfate donor, 3'-phosphoadenosine-5'-phosphosulfate, yet SULT4A1 is classified as a SULT superfamily member based on sequence and structural similarities to the other SULTs. In this study, transcription activator-like effector nucleases were used to generate heritable mutations in the SULT4A1 gene of zebrafish. The mutation (SULT4A1(Δ8)) consists of an 8-nucleotide deletion within the second exon of the gene, resulting in a frameshift mutation and premature stop codon after 132 AA. During early adulthood, casual observations were made that mutant zebrafish were exhibiting excessively sedentary behavior during the day. These observations were inconsistent with published reports on activity in zebrafish that are largely diurnal organisms and are highly active during the day. Thus, a decrease in activity during the day represents an abnormal behavior and warranted further systematic analysis. EthoVision video tracking software was used to monitor activity levels in wild-type (WT) and SULT4A1(Δ8/Δ8) fish over 48 hours of a normal light/dark cycle. SULT4A1(Δ8/Δ8) fish were shown to exhibit increased inactivity bout length and frequency as well as a general decrease in daytime activity levels when compared with their WT counterparts.


Asunto(s)
Mutación del Sistema de Lectura/genética , Actividad Motora/genética , Sulfotransferasas/genética , Sulfotransferasas/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Secuencia de Aminoácidos , Animales , Ansiedad/genética , Ansiedad/psicología , Secuencia de Bases , Desoxirribonucleasas/administración & dosificación , Desoxirribonucleasas/farmacología , Embrión no Mamífero , Exones , Microinyecciones , Datos de Secuencia Molecular , Mutación , Conducta Social
11.
PLoS One ; 9(12): e114632, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25503746

RESUMEN

With the goal to generate and characterize the phenotypes of null alleles in all genes within an organism and the recent advances in custom nucleases, genome editing limitations have moved from mutation generation to mutation detection. We previously demonstrated that High Resolution Melting (HRM) analysis is a rapid and efficient means of genotyping known zebrafish mutants. Here we establish optimized conditions for HRM based detection of novel mutant alleles. Using these conditions, we demonstrate that HRM is highly efficient at mutation detection across multiple genome editing platforms (ZFNs, TALENs, and CRISPRs); we observed nuclease generated HRM positive targeting in 1 of 6 (16%) open pool derived ZFNs, 14 of 23 (60%) TALENs, and 58 of 77 (75%) CRISPR nucleases. Successful targeting, based on HRM of G0 embryos correlates well with successful germline transmission (46 of 47 nucleases); yet, surprisingly mutations in the somatic tail DNA weakly correlate with mutations in the germline F1 progeny DNA. This suggests that analysis of G0 tail DNA is a good indicator of the efficiency of the nuclease, but not necessarily a good indicator of germline alleles that will be present in the F1s. However, we demonstrate that small amplicon HRM curve profiles of F1 progeny DNA can be used to differentiate between specific mutant alleles, facilitating rare allele identification and isolation; and that HRM is a powerful technique for screening possible off-target mutations that may be generated by the nucleases. Our data suggest that micro-homology based alternative NHEJ repair is primarily utilized in the generation of CRISPR mutant alleles and allows us to predict likelihood of generating a null allele. Lastly, we demonstrate that HRM can be used to quickly distinguish genotype-phenotype correlations within F1 embryos derived from G0 intercrosses. Together these data indicate that custom nucleases, in conjunction with the ease and speed of HRM, will facilitate future high-throughput mutation generation and analysis needed to establish mutants in all genes of an organism.


Asunto(s)
Ingeniería Genética/métodos , Genómica , Técnicas de Genotipaje/métodos , Fenotipo , Temperatura de Transición , Pez Cebra/genética , Animales , Composición de Base , Secuencia de Bases , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Daño del ADN , Análisis Mutacional de ADN , Desoxirribonucleasas/química , Desoxirribonucleasas/metabolismo , Hibridación Genética , Mutación , Desnaturalización de Ácido Nucleico , Dedos de Zinc
12.
Drug Metab Dispos ; 42(5): 947-53, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24553382

RESUMEN

Sulfotransferase (SULT) 4A1 is an orphan enzyme that shares distinct structure and sequence similarities with other cytosolic SULTs. SULT4A1 is primarily expressed in neuronal tissue and is also the most conserved SULT, having been identified in every vertebrate investigated to date. Certain haplotypes of the SULT4A1 gene are correlated with higher baseline psychopathology in schizophrenic patients, but no substrate or function for SULT4A1 has yet been identified despite its high level of sequence conservation. In this study, deep RNA sequencing was used to search for alterations in gene expression in 72-hour postfertilization zebrafish larvae following transient SULT4A1 knockdown (KD) utilizing splice blocking morpholino oligonucleotides. This study demonstrates that transient inhibition of SULT4A1 expression in developing zebrafish larvae results in the up-regulation of several genes involved in phototransduction. SULT4A1 KD was verified by immunoblot analysis and quantitative real-time polymerase chain reaction (qPCR). Gene regulation changes identified by deep RNA sequencing were validated by qPCR. This study is the first identification of a cellular process whose regulation appears to be associated with SULT4A1 expression.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Fototransducción/genética , Sulfotransferasas/fisiología , Transcriptoma , Proteínas de Pez Cebra/fisiología , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/embriología , Encéfalo/metabolismo , Ojo/embriología , Ojo/metabolismo , Fertilización , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Larva , Datos de Secuencia Molecular , Morfolinos/farmacología , ARN/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ARN , Homología de Secuencia de Aminoácido , Sulfotransferasas/genética , Regulación hacia Arriba , Pez Cebra/embriología , Proteínas de Pez Cebra/genética
13.
Dis Model Mech ; 3(1-2): 45-56, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20075382

RESUMEN

Li-Fraumeni syndrome (LFS) is a highly penetrant, autosomal dominant, human familial cancer predisposition. Although a key role for the tumor suppressor p53 has been implicated in LFS, the genetic and cellular mechanisms underpinning this disease remain unknown. Therefore, modeling LFS in a vertebrate system that is accessible to both large-scale genetic screens and in vivo cell biological studies will facilitate the in vivo dissection of disease mechanisms, help identify candidate genes, and spur the discovery of therapeutic compounds. Here, we describe a forward genetic screen in zebrafish embryos that was used to identify LFS candidate genes, which yielded a p53 mutant (p53(I166T)) that as an adult develops tumors, predominantly sarcomas, with 100% penetrance. As in humans with LFS, tumors arise in heterozygotes and display loss of heterozygosity (LOH). This report of LOH indicates that Knudson's two-hit hypothesis, a hallmark of human autosomal dominant cancer syndromes, can be modeled in zebrafish. Furthermore, as with some LFS mutations, the zebrafish p53(I166T) allele is a loss-of-function allele with dominant-negative activity in vivo. Additionally, we demonstrate that the p53 regulatory pathway, including Mdm2 regulation, is evolutionarily conserved in zebrafish, providing a bona fide biological context in which to systematically uncover novel modifier genes and therapeutic agents for human LFS.


Asunto(s)
Síndrome de Li-Fraumeni/genética , Modelos Genéticos , Pez Cebra/genética , Alelos , Animales , Apoptosis/efectos de la radiación , Daño del ADN , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Genes Dominantes/genética , Pruebas Genéticas , Heterocigoto , Pérdida de Heterocigocidad/genética , Mutación/genética , Neoplasias/genética , Neoplasias/patología , Estabilidad Proteica/efectos de la radiación , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Radiación Ionizante , Transducción de Señal/efectos de la radiación , Activación Transcripcional/genética , Activación Transcripcional/efectos de la radiación , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo
14.
Dev Dyn ; 238(12): 3168-74, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19890916

RESUMEN

In order to facilitate high throughput genotyping of zebrafish, we have developed a novel technique that uses High Resolution Melting Analysis (HRMA) to distinguish wild-type, heterozygous mutants and homogyzous mutants. This one hour technique removes the need for restriction enzymes and agarose gels. The generated melting curve profiles are sensitive enough to detect non-specific PCR products. We have been able to reliably genotype three classes of mutations in zebrafish, including point mutants, apc(hu745) (apc(mcr)), and p53(zy7) (p53(I166T)), a small deletion mutant (bap28(y75)) and a retroviral insertion mutant (wdr43(hi821a)). This technique can genotype individual zebrafish embryos and adults (by tail-clip) and is applicable to other model organisms.


Asunto(s)
Análisis Citogenético/métodos , Análisis Mutacional de ADN/métodos , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Eficiencia , Embrión no Mamífero , Eliminación de Gen , Genotipo , Datos de Secuencia Molecular , Mutagénesis Insercional , Proteínas Mutantes/análisis , Proteínas Mutantes/genética , Polimorfismo de Nucleótido Simple , Retroviridae/genética , Retroviridae/fisiología , Factores de Tiempo , Pez Cebra/embriología , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismo
15.
Cancer Res ; 68(21): 8968-75, 2008 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-18974141

RESUMEN

Ewing's sarcoma is characterized by the t(11;22)(q24:q12) reciprocal translocation. To study the effects of the fusion gene EWS-FLI1 on development and tumor formation, a transgenic mouse model was created. A strategy of conditional expression was used to limit the potentially deleterious effects of EWS-FLI1 to certain tissues. In the absence of Cre recombinase, EWS-FLI1 was not expressed in the EWS-FLI1 transgenic mice, and they had a normal phenotype. When crossed to the Prx1-Cre transgenic mouse, which expresses Cre recombinase in the primitive mesenchymal cells of the embryonic limb bud, the EF mice were noted to have a number of developmental defects of the limbs. These included shortening of the limbs, muscle atrophy, cartilage dysplasia, and immature bone. By itself, EWS-FLI1 did not induce the formation of tumors in the EF transgenic mice. However, in the setting of p53 deletion, EWS-FLI1 accelerated the formation of sarcomas from a median time of 50 to 21 weeks. Furthermore, EWS-FLI1 altered the type of tumor that formed. Conditional deletion of p53 in mesenchymal cells (Prx1-Cre p53(lox/lox)) produced osteosarcomas as the predominant tumor. The presence of EWS-FLI1 shifted the tumor phenotype to a poorly differentiated sarcoma. The results taken together suggest that EWS-FLI1 inhibits normal limb development and accelerates the formation of poorly differentiated sarcomas.


Asunto(s)
Deformidades Congénitas de las Extremidades/genética , Proteínas de Fusión Oncogénica/fisiología , Sarcoma Experimental/patología , Factores de Transcripción/fisiología , Animales , Secuencia de Bases , Western Blotting , División Celular , Cartilla de ADN , Inmunohistoquímica , Ratones , Ratones Transgénicos , Proteínas de Fusión Oncogénica/genética , Proteína Proto-Oncogénica c-fli-1 , Proteína EWS de Unión a ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sarcoma Experimental/genética , Factores de Transcripción/genética
16.
Dev Dyn ; 236(11): 3088-99, 2007 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17937395

RESUMEN

Transgenesis is an important tool for assessing gene function. In zebrafish, transgenesis has suffered from three problems: the labor of building complex expression constructs using conventional subcloning; low transgenesis efficiency, leading to mosaicism in transient transgenics and infrequent germline incorporation; and difficulty in identifying germline integrations unless using a fluorescent marker transgene. The Tol2kit system uses site-specific recombination-based cloning (multisite Gateway technology) to allow quick, modular assembly of [promoter]-[coding sequence]-[3' tag] constructs in a Tol2 transposon backbone. It includes a destination vector with a cmlc2:EGFP (enhanced green fluorescent protein) transgenesis marker and a variety of widely useful entry clones, including hsp70 and beta-actin promoters; cytoplasmic, nuclear, and membrane-localized fluorescent proteins; and internal ribosome entry sequence-driven EGFP cassettes for bicistronic expression. The Tol2kit greatly facilitates zebrafish transgenesis, simplifies the sharing of clones, and enables large-scale projects testing the functions of libraries of regulatory or coding sequences.


Asunto(s)
Animales Modificados Genéticamente , Clonación Molecular/métodos , Elementos Transponibles de ADN , ADN Recombinante/genética , Técnicas de Transferencia de Gen , Pez Cebra/genética , Animales , Técnicas Genéticas , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Plásmidos/genética , Recombinación Genética , Transposasas/metabolismo , Pez Cebra/metabolismo
17.
Mol Cell Biol ; 26(1): 192-8, 2006 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-16354690

RESUMEN

The function of the p53 tumor suppressor to inhibit proliferation or initiate apoptosis is often abrogated in tumor cells. Mdm2 and its homolog, Mdm4, are critical inhibitors of p53 that are often overexpressed in human tumors. In mice, loss of Mdm2 or Mdm4 leads to embryonic lethal phenotypes that are completely rescued by concomitant loss of p53. To examine the role of Mdm2 and Mdm4 in a temporal and tissue-specific manner and to determine the relationships of these inhibitors to each other, we generated conditional alleles. We deleted Mdm2 and Mdm4 in cardiomyocytes, since proliferation and apoptosis are important processes in heart development. Mice lacking Mdm2 in the heart were embryonic lethal and showed defects at the time recombination occurred. A critical number of cardiomyocytes were lost by embryonic day 13.5, resulting in heart failure. This phenotype was completely rescued by deletion of p53. Mice lacking Mdm4 in the heart were born at the correct ratio and appeared to be normal. Our studies provide the first direct evidence that Mdm2 can function in the absence of Mdm4 to regulate p53 activity in a tissue-specific manner. Moreover, Mdm4 cannot compensate for the loss of Mdm2 in heart development.


Asunto(s)
Cardiopatías Congénitas/genética , Corazón/embriología , Proteínas Proto-Oncogénicas c-mdm2/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/fisiología , Alelos , Animales , Apoptosis , Proliferación Celular , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , Eliminación de Gen , Genes Letales , Cardiopatías Congénitas/embriología , Ratones , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteína p53 Supresora de Tumor/genética , Ubiquitina-Proteína Ligasas/genética
18.
Mol Endocrinol ; 19(6): 1646-53, 2005 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15831521

RESUMEN

The constitutive androstane receptor (CAR, NR1I3) is a central regulator of xenobiotic metabolism. CAR activation induces hepatic expression of detoxification enzymes and transporters and increases liver size. Here we show that CAR-mediated hepatomegaly is a transient, adaptive response to acute xenobiotic stress. In contrast, chronic CAR activation results in hepatocarcinogenesis. In both acute and chronic xenobiotic responses, hepatocyte DNA replication is increased and apoptosis is decreased. These effects are absent in CAR null mice, which are completely resistant to tumorigenic effects of chronic xenobiotic stress. In the acute response, direct up-regulation of Mdm2 expression by CAR contributes to both increased DNA replication and inhibition of p53-mediated apoptosis. These results demonstrate an essential role for CAR in regulating both liver homeostasis and tumorigenesis in response to xenobiotic stresses, and they also identify a specific molecular mechanism linking chronic environmental stress and tumor formation.


Asunto(s)
Hepatomegalia/patología , Neoplasias Hepáticas/patología , Receptores Citoplasmáticos y Nucleares/fisiología , Factores de Transcripción/fisiología , Animales , Apoptosis , Proliferación Celular , Inmunoprecipitación de Cromatina , ADN/metabolismo , Fragmentación del ADN , Citometría de Flujo , Células HeLa , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/etiología , Ratones , Ratones Transgénicos , Proteínas Nucleares/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2 , ARN/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Tiempo , Factores de Transcripción/metabolismo , Transfección , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba , Xenobióticos/química
19.
Cell ; 119(6): 861-72, 2004 Dec 17.
Artículo en Inglés | MEDLINE | ID: mdl-15607981

RESUMEN

Individuals with Li-Fraumeni syndrome carry inherited mutations in the p53 tumor suppressor gene and are predisposed to tumor development. To examine the mechanistic nature of these p53 missense mutations, we generated mice harboring a G-to-A substitution at nucleotide 515 of p53 (p53+/515A) corresponding to the p53R175H hot spot mutation in human cancers. Although p53+/515A mice display a similar tumor spectrum and survival curve as p53+/- mice, tumors from p53+/515A mice metastasized with high frequency. Correspondingly, the embryonic fibroblasts from the p53515A/515A mutant mice displayed enhanced cell proliferation, DNA synthesis, and transformation potential. The disruption of p63 and p73 in p53-/- cells increased transformation capacity and reinitiated DNA synthesis to levels observed in p53515A/515A cells. Additionally, p63 and p73 were functionally inactivated in p53515A cells. These results provide in vivo validation for the gain-of-function properties of certain p53 missense mutations and suggest a mechanistic basis for these phenotypes.


Asunto(s)
Transformación Celular Neoplásica/genética , Fibroblastos/metabolismo , Genes p53/genética , Síndrome de Li-Fraumeni/genética , Neoplasias/genética , Animales , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Células Cultivadas , Replicación del ADN/genética , Replicación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Fibroblastos/citología , Genes Supresores de Tumor , Genes p53/fisiología , Síndrome de Li-Fraumeni/metabolismo , Síndrome de Li-Fraumeni/patología , Ratones , Ratones Transgénicos , Mutación/genética , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Ratas , Transactivadores/metabolismo , Proteína Tumoral p73 , Proteínas Supresoras de Tumor
20.
Oncogene ; 23(46): 7644-50, 2004 Oct 07.
Artículo en Inglés | MEDLINE | ID: mdl-15361844

RESUMEN

Mdm2 and its homolog Mdm4 inhibit the function of the tumor suppressor p53. Targeted disruption of either mdm2 or mdm4 genes in mice results in embryonic lethality that is completely rescued by concomitant deletion of p53, suggesting that deletion of negative regulators of p53 results in a constitutively active p53. Thus, these mouse models offer a unique in vivo system to assay the functional significance of different p53 modifications. Phosphorylation of serine 389 in murine p53 occurs specifically after ultraviolet-light-induced DNA damage, and phosphorylation of this site enhances p53 activity both in vitro and in vivo. Recently, mice with a serine to alanine substitution at serine 389 (p53S389A) in the endogenous p53 locus were generated. To examine the in vivo significance of serine 389 phosphorylation during embryogenesis, we crossed these mutant mice to mice lacking mdm2 or mdm4. The p53S389A allele did not alter the embryonic lethality of mdm2 or mdm4. Additional crosses to assay the effect of one p53S389A allele with a p53 null allele also did not rescue the lethal phenotypes. In conclusion, the phenotypes due to loss of mdm2 or mdm4 were not even partially rescued by p53S389A, suggesting that p53S389A is functionally wild type during embryogenesis.


Asunto(s)
Proteínas Nucleares/deficiencia , Proteínas Proto-Oncogénicas/deficiencia , Serina , Proteína p53 Supresora de Tumor/genética , Animales , Muerte Fetal/genética , Fibroblastos/citología , Fibroblastos/fisiología , Ratones , Ratones Noqueados , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-mdm2 , Transcripción Genética/efectos de la radiación , Ubiquitina-Proteína Ligasas , Rayos Ultravioleta
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...