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Chondroitin/dermatan sulfate (CS/DS) proteoglycans are indispensable for animal development and homeostasis but the large number of enzymes involved in their biosynthesis have made CS/DS function a challenging problem to study genetically. In our study, we generated loss-of-function alleles in zebrafish genes encoding CS/DS biosynthetic enzymes and characterized the effect on development in single and double mutants. Homozygous mutants in chsy1, csgalnact1a, csgalnat2, chpfa, ust and chst7, respectively, develop to adults. However, csgalnact1a-/- fish develop distinct craniofacial defects while the chsy1-/- skeletal phenotype is milder and the remaining mutants display no gross morphological abnormalities. These results suggest a high redundancy for the CS/DS biosynthetic enzymes and to further reduce CS/DS biosynthesis we combined mutant alleles. The craniofacial phenotype is further enhanced in csgalnact1a-/-;chsy1-/- adults and csgalnact1a-/-;csgalnact2-/- larvae. While csgalnact1a-/-;csgalnact2-/- was the most affected allele combination in our study, CS/DS is still not completely abolished. Transcriptome analysis of chsy1-/-, csgalnact1a-/- and csgalnact1a-/-;csgalnact2-/- larvae revealed that the expression had changed in a similar way in the three mutant lines but no differential expression was found in any of fifty GAG biosynthesis enzymes identified. Thus, zebrafish larvae do not increase transcription of GAG biosynthesis genes as a consequence of decreased CS/DS biosynthesis. The new zebrafish lines develop phenotypes similar to clinical characteristics of several human congenital disorders making the mutants potentially useful to study disease mechanisms and treatment.
Assuntos
Dermatan Sulfato , Peixe-Zebra , Animais , Sulfatos de Condroitina/metabolismo , Dermatan Sulfato/genética , Dermatan Sulfato/metabolismo , Glicosiltransferases/genética , Fenótipo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
The coherent perfect absorption (CPA) occurring in the graphene sheet suspended in air can be utilized to develop an ultrathin, ultra-broadband absorber working in the frequency range from a few hertz (Hz) to terahertz (THz) with perfect absorption. A graphene sheet is studied to induce the CPA to cover radio, microwave and lower THz frequency ranges. A graphene resonator able to provide the surface plasmon resonance (SPR) is combined with the graphene sheet to provide CPA at either side of a thin dielectric layer forms metamaterial structure with the cavity and enhances the absorption bandwidth in the THz region by creating a resonance near quasi-CPA frequency. A dielectric silicon resonator is embedded in the structure, which creates dipolar resonances between the resonances obtained by the formed cavity between the graphene sheet and resonator. This enhances the absorption level in the THz region. The absorption bandwidth is further enhanced to 7 THz by including a graphene disc at the top of the silicon resonator. Thus, the multiple multi-order resonances occurring in the silicon dielectric and SPR of graphene resonators are merged with the phenomena of CPA occurring in the graphene sheets to extend the CPA bandwidth in the THz regime. The doping level of graphene or its tunable Fermi energy based on the applied DC electric field provides the tunability in the total obtained absorption bandwidth. The symmetric structure provides polarization-insensitive behavior with an allowed incident angle of more than 45° with more than 90% absorption.
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A terahertz (THz) antenna with tunable filtering is designed and numerically studied. A slotted monopole radiator with defected ground structure is used for operating with wideband response with an impedance bandwidth in the range of 3.80-11.98 THz for S 11≤-10d B. A slot is engraved in the radiator for obtaining the filtering characteristics in the antenna response. By varying its chemical potential, the frequency band 5.05-6.69 THz with graphene material can provide the tunability in the frequency and bandwidth of the filtered band in the antenna response. Furthermore, the antenna can provide radiation efficiency of more than 90% and gain with a peak value of 6.6 dBi in the passband.
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Aminoacyl-tRNA synthetases (ARSs) are essential enzymes for faithful assignment of amino acids to their cognate tRNA. Variants in ARS genes are frequently associated with clinically heterogeneous phenotypes in humans and follow both autosomal dominant or recessive inheritance patterns in many instances. Variants in tryptophanyl-tRNA synthetase 1 (WARS1) cause autosomal dominantly inherited distal hereditary motor neuropathy and Charcot-Marie-Tooth disease. Presently, only one family with biallelic WARS1 variants has been described. We present three affected individuals from two families with biallelic variants (p.Met1? and p.(Asp419Asn)) in WARS1, showing varying severities of developmental delay and intellectual disability. Hearing impairment and microcephaly, as well as abnormalities of the brain, skeletal system, movement/gait, and behavior were variable features. Phenotyping of knocked down wars-1 in a Caenorhabditis elegans model showed depletion is associated with defects in germ cell development. A wars1 knockout vertebrate model recapitulates the human clinical phenotypes, confirms variant pathogenicity, and uncovers evidence implicating the p.Met1? variant as potentially impacting an exon critical for normal hearing. Together, our findings provide consolidating evidence for biallelic disruption of WARS1 as causal for an autosomal recessive neurodevelopmental syndrome and present a vertebrate model that recapitulates key phenotypes observed in patients.
Assuntos
Aminoacil-tRNA Sintetases , Doença de Charcot-Marie-Tooth , Triptofano-tRNA Ligase , Aminoacil-tRNA Sintetases/genética , Doença de Charcot-Marie-Tooth/genética , Éxons , Humanos , Mutação , Linhagem , RNA de Transferência/genética , Síndrome , Triptofano-tRNA Ligase/genéticaRESUMO
Aminoacylation of transfer RNA (tRNA) is a key step in protein biosynthesis, carried out by highly specific aminoacyl-tRNA synthetases (ARSs). ARSs have been implicated in autosomal dominant and autosomal recessive human disorders. Autosomal dominant variants in tryptophanyl-tRNA synthetase 1 (WARS1) are known to cause distal hereditary motor neuropathy and Charcot-Marie-Tooth disease, but a recessively inherited phenotype is yet to be clearly defined. Seryl-tRNA synthetase 1 (SARS1) has rarely been implicated in an autosomal recessive developmental disorder. Here, we report five individuals with biallelic missense variants in WARS1 or SARS1, who presented with an overlapping phenotype of microcephaly, developmental delay, intellectual disability, and brain anomalies. Structural mapping showed that the SARS1 variant is located directly within the enzyme's active site, most likely diminishing activity, while the WARS1 variant is located in the N-terminal domain. We further characterize the identified WARS1 variant by showing that it negatively impacts protein abundance and is unable to rescue the phenotype of a CRISPR/Cas9 wars1 knockout zebrafish model. In summary, we describe two overlapping autosomal recessive syndromes caused by variants in WARS1 and SARS1, present functional insights into the pathogenesis of the WARS1-related syndrome and define an emerging disease spectrum: ARS-related developmental disorders with or without microcephaly.
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Aminoacil-tRNA Sintetases , Doença de Charcot-Marie-Tooth , Microcefalia , Triptofano-tRNA Ligase , Animais , Humanos , Aminoacil-tRNA Sintetases/genética , Doença de Charcot-Marie-Tooth/genética , Ligases , Microcefalia/genética , Microcefalia/patologia , RNA de Transferência , Triptofano-tRNA Ligase/genética , Peixe-Zebra/genéticaRESUMO
Mutations in the transcription factor GATA2 cause lymphedema. GATA2 is necessary for the development of lymphatic valves and lymphovenous valves, and for the patterning of lymphatic vessels. Here, we report that GATA2 is not necessary for valvular endothelial cell (VEC) differentiation. Instead, GATA2 is required for VEC maintenance and morphogenesis. GATA2 is also necessary for the expression of the cell junction molecules VE-cadherin and claudin 5 in lymphatic vessels. We identified miR-126 as a target of GATA2, and miR-126-/- embryos recapitulate the phenotypes of mice lacking GATA2. Primary human lymphatic endothelial cells (HLECs) lacking GATA2 (HLECΔGATA2) have altered expression of claudin 5 and VE-cadherin, and blocking miR-126 activity in HLECs phenocopies these changes in expression. Importantly, overexpression of miR-126 in HLECΔGATA2 significantly rescues the cell junction defects. Thus, our work defines a new mechanism of GATA2 activity and uncovers miR-126 as a novel regulator of mammalian lymphatic vascular development.
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Células Endoteliais/metabolismo , Fator de Transcrição GATA2/metabolismo , MicroRNAs/metabolismo , Mutação , Angiopoietina-2/metabolismo , Animais , Sistemas CRISPR-Cas , Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular , Linhagem Celular , Claudina-5/metabolismo , Família de Proteínas EGF/metabolismo , Endotélio Vascular/metabolismo , Feminino , Deleção de Genes , Humanos , Vasos Linfáticos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA-SeqRESUMO
PURPOSE: The mediator (MED) multisubunit-complex modulates the activity of the transcriptional machinery, and genetic defects in different MED subunits (17, 20, 27) have been implicated in neurologic diseases. In this study, we identified a recurrent homozygous variant in MED11 (c.325C>T; p.Arg109Ter) in 7 affected individuals from 5 unrelated families. METHODS: To investigate the genetic cause of the disease, exome or genome sequencing were performed in 5 unrelated families identified via different research networks and Matchmaker Exchange. Deep clinical and brain imaging evaluations were performed by clinical pediatric neurologists and neuroradiologists. The functional effect of the candidate variant on both MED11 RNA and protein was assessed using reverse transcriptase polymerase chain reaction and western blotting using fibroblast cell lines derived from 1 affected individual and controls and through computational approaches. Knockouts in zebrafish were generated using clustered regularly interspaced short palindromic repeats/Cas9. RESULTS: The disease was characterized by microcephaly, profound neurodevelopmental impairment, exaggerated startle response, myoclonic seizures, progressive widespread neurodegeneration, and premature death. Functional studies on patient-derived fibroblasts did not show a loss of protein function but rather disruption of the C-terminal of MED11, likely impairing binding to other MED subunits. A zebrafish knockout model recapitulates key clinical phenotypes. CONCLUSION: Loss of the C-terminal of MED subunit 11 may affect its binding efficiency to other MED subunits, thus implicating the MED-complex stability in brain development and neurodegeneration.
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Complexo Mediador , Microcefalia , Doenças Neurodegenerativas , Animais , Humanos , Homozigoto , Complexo Mediador/genética , Microcefalia/genética , Doenças Neurodegenerativas/genética , RNA , Peixe-Zebra/genéticaRESUMO
A circularly polarized (CP) multi-input multioutput (MIMO) dielectric resonator (DR) antenna (DRA) with compact size and four ports is implemented. CP radiation was achieved using the deformed DR geometry excited with aperture coupled feeding. A CPDRA with a single and two ports is investigated. The defected ground structure (DGS) was incorporated into the antenna for improving the isolation between the ports. The DGS was incorporated in such a way that the required phase difference between the generated orthogonal degenerate modes is preserved. This concept could be utilized in implementing a compact four-port CP antenna. The MIMO antenna provides a 10 dB impedance bandwidth of 38% (8.5-12.5 GHz) and a 3 dB AR bandwidth of 9.32% (9.2-10.1 GHz). The gain of the implemented antenna was around 6 dBi in the band where CP radiation was achieved. The MIMO performance parameters were calculated, and their values remained within the acceptable limits. The implemented antenna could suitably be used in X-band applications.
Assuntos
Tecnologia sem Fio , Impedância Elétrica , Desenho de EquipamentoRESUMO
Deafness, the most frequent sensory deficit in humans, is extremely heterogeneous with hundreds of genes involved. Clinical and genetic analyses of an extended consanguineous family with pre-lingual, moderate-to-profound autosomal recessive sensorineural hearing loss, allowed us to identify CLRN2, encoding a tetraspan protein, as a new deafness gene. Homozygosity mapping followed by exome sequencing identified a 14.96 Mb locus on chromosome 4p15.32p15.1 containing a likely pathogenic missense variant in CLRN2 (c.494C > A, NM_001079827.2) segregating with the disease. Using in vitro RNA splicing analysis, we show that the CLRN2 c.494C > A variant leads to two events: (1) the substitution of a highly conserved threonine (uncharged amino acid) to lysine (charged amino acid) at position 165, p.(Thr165Lys), and (2) aberrant splicing, with the retention of intron 2 resulting in a stop codon after 26 additional amino acids, p.(Gly146Lysfs*26). Expression studies and phenotyping of newly produced zebrafish and mouse models deficient for clarin 2 further confirm that clarin 2, expressed in the inner ear hair cells, is essential for normal organization and maintenance of the auditory hair bundles, and for hearing function. Together, our findings identify CLRN2 as a new deafness gene, which will impact future diagnosis and treatment for deaf patients.
Assuntos
Substituição de Aminoácidos , Cromossomos Humanos Par 4/química , Células Ciliadas Auditivas Internas/metabolismo , Perda Auditiva Neurossensorial/genética , Proteínas de Membrana/genética , Mutação Puntual , Tetraspaninas/genética , Adulto , Alelos , Animais , Sequência de Bases , Mapeamento Cromossômico , Consanguinidade , Feminino , Expressão Gênica , Genes Recessivos , Células Ciliadas Auditivas Internas/patologia , Perda Auditiva Neurossensorial/metabolismo , Perda Auditiva Neurossensorial/patologia , Humanos , Masculino , Proteínas de Membrana/deficiência , Camundongos , Linhagem , Tetraspaninas/deficiência , Sequenciamento do Exoma , Peixe-ZebraRESUMO
A reconfigurable graphene antenna is designed and numerically analyzed. The antenna structure contains a radiating graphene patch and non-radiating graphene ring. The operating frequency of the antenna can be tuned by applying the gate voltage on the graphene patch. This proximity coupled antenna structure operates with the [Formula: see text] mode. In this configuration, the field is distributed in the form of horizontal magnetic dipole. The application of gate voltage at the periphery of graphene ring converts the field configuration to the form of the vertical electric dipole. Thus, the mode of operation of the antenna is changed to [Formula: see text] Consequently, the radiation pattern of the antenna can be steered in different directions as per the location of the applied gate voltage at the periphery of the graphene ring.
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The Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) plays a critical role in the specification of founder cells (FCs) in the Drosophila visceral mesoderm (VM) during embryogenesis. Reporter gene and CRISPR/Cas9 deletion analysis reveals enhancer regions in and upstream of the Alk locus that influence tissue-specific expression in the amnioserosa (AS), the VM and the epidermis. By performing high throughput yeast one-hybrid screens (Y1H) with a library of Drosophila transcription factors (TFs) we identify Odd-paired (Opa), the Drosophila homologue of the vertebrate Zic family of TFs, as a novel regulator of embryonic Alk expression. Further characterization identifies evolutionarily conserved Opa-binding cis-regulatory motifs in one of the Alk associated enhancer elements. Employing Alk reporter lines as well as CRISPR/Cas9-mediated removal of regulatory elements in the Alk locus, we show modulation of Alk expression by Opa in the embryonic AS, epidermis and VM. In addition, we identify enhancer elements that integrate input from additional TFs, such as Binou (Bin) and Bagpipe (Bap), to regulate VM expression of Alk in a combinatorial manner. Taken together, our data show that the Opa zinc finger TF is a novel regulator of embryonic Alk expression.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Receptores Proteína Tirosina Quinases/genética , Fatores de Transcrição/genética , Complexo 1 de Proteínas Adaptadoras/genética , Complexo 1 de Proteínas Adaptadoras/metabolismo , Subunidades beta do Complexo de Proteínas Adaptadoras/genética , Subunidades beta do Complexo de Proteínas Adaptadoras/metabolismo , Quinase do Linfoma Anaplásico , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Sistemas CRISPR-Cas , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Embrião não Mamífero , Elementos Facilitadores Genéticos , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Proteínas de Homeodomínio/metabolismo , Regiões Promotoras Genéticas , Receptores Proteína Tirosina Quinases/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The use of CRISPR/Cas9 as a genome-editing tool in various model organisms has radically changed targeted mutagenesis. Here, we present a high-throughput targeted mutagenesis pipeline using CRISPR/Cas9 technology in zebrafish that will make possible both saturation mutagenesis of the genome and large-scale phenotyping efforts. We describe a cloning-free single-guide RNA (sgRNA) synthesis, coupled with streamlined mutant identification methods utilizing fluorescent PCR and multiplexed, high-throughput sequencing. We report germline transmission data from 162 loci targeting 83 genes in the zebrafish genome, in which we obtained a 99% success rate for generating mutations and an average germline transmission rate of 28%. We verified 678 unique alleles from 58 genes by high-throughput sequencing. We demonstrate that our method can be used for efficient multiplexed gene targeting. We also demonstrate that phenotyping can be done in the F1 generation by inbreeding two injected founder fish, significantly reducing animal husbandry and time. This study compares germline transmission data from CRISPR/Cas9 with those of TALENs and ZFNs and shows that efficiency of CRISPR/Cas9 is sixfold more efficient than other techniques. We show that the majority of published "rules" for efficient sgRNA design do not effectively predict germline transmission rates in zebrafish, with the exception of a GG or GA dinucleotide genomic match at the 5' end of the sgRNA. Finally, we show that predicted off-target mutagenesis is of low concern for in vivo genetic studies.
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Sistemas CRISPR-Cas , Marcação de Genes , Ensaios de Triagem em Larga Escala , Fenótipo , Alelos , Animais , Técnicas de Inativação de Genes , Marcação de Genes/métodos , Estudo de Associação Genômica Ampla , Genômica , Células Germinativas/imunologia , Humanos , Mutagênese , Locos de Características Quantitativas , RNA Guia de Cinetoplastídeos/genética , Deleção de Sequência , Peixe-ZebraRESUMO
CRISPRz (http://research.nhgri.nih.gov/CRISPRz/) is a database of CRISPR/Cas9 target sequences that have been experimentally validated in zebrafish. Programmable RNA-guided CRISPR/Cas9 has recently emerged as a simple and efficient genome editing method in various cell types and organisms, including zebrafish. Because the technique is so easy and efficient in zebrafish, the most valuable asset is no longer a mutated fish (which has distribution challenges), but rather a CRISPR/Cas9 target sequence to the gene confirmed to have high mutagenic efficiency. With a highly active CRISPR target, a mutant fish can be quickly replicated in any genetic background anywhere in the world. However, sgRNA's vary widely in their activity and models for predicting target activity are imperfect. Thus, it is very useful to collect in one place validated CRISPR target sequences with their relative mutagenic activities. A researcher could then select a target of interest in the database with an expected activity. Here, we report the development of CRISPRz, a database of validated zebrafish CRISPR target sites collected from published sources, as well as from our own in-house large-scale mutagenesis project. CRISPRz can be searched using multiple inputs such as ZFIN IDs, accession number, UniGene ID, or gene symbols from zebrafish, human and mouse.
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Sistemas CRISPR-Cas , Bases de Dados Genéticas , RNA , Peixe-Zebra/genética , Animais , Marcação de Genes , Humanos , Camundongos , Mutagênese , Peixe-Zebra/embriologiaRESUMO
Diverse cellular processes depend on endocytosis, intracellular vesicle trafficking, sorting and exocytosis, processes regulated post-transcriptionally by modifications such as phosphorylation and ubiquitylation. In addition to sorting to the lysosome, cargo is recycled to the plasma membrane via recycling endosomes. Here, we describe a role of the goliath gene family of protease-associated (PA) domain E3 ligases in regulating recycling endosome trafficking. The two Drosophila members of this family--Goliath and Godzilla(CG10277)--are located on endosomes, and both ectopic expression and loss-of-function lead to the accumulation of Rab5-positive giant endosomes. Furthermore, the human homologue RNF167 exhibits similar behaviour. We show that the soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) protein VAMP3 is a target of these ubiquitin ligases, and that recycling endosome trafficking is abrogated in response to their activity. Furthermore, mutation of the Godzilla ubiquitylation target lysines on VAMP3 abrogates the formation of enlarged endosomes induced by either Godzilla or RNF167. Thus, Goliath ubiquitin ligases play a novel role in regulating recycling endosome trafficking via ubiquitylation of the VAMP3 SNARE protein.
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Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Endossomos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , Proteína 3 Associada à Membrana da Vesícula/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Endossomos/genética , Células HEK293 , Humanos , Ubiquitina-Proteína Ligases/genética , Proteína 3 Associada à Membrana da Vesícula/genéticaRESUMO
CRISPR/Cas9 has emerged as a versatile genome-engineering tool that relies on a single guide RNA (sgRNA) and the Cas9 enzyme for genome editing. Simple, fast and economical methods to generate sgRNAs have made targeted mutagenesis routine in cultured cells, mice, zebrafish and other model systems. Pre-screening of sgRNAs for target efficacy is desirable both for successful mutagenesis and minimizing wasted animal husbandry on targets with poor activity. Here, we describe an easy, quick and cost-effective fluorescent polymerase chain reaction (PCR)-based method, CRISPR Somatic Tissue Activity Test (CRISPR-STAT), to determine target-specific efficiency of sgRNA. As a proof of principle, we validated our method using 28 sgRNAs with known and varied levels of germline transmission efficiency in zebrafish by analysis of their somatic activity in injected embryos. Our data revealed a strong positive correlation between the fluorescent PCR profiles of the injected embryos and the germline transmission efficiency. Furthermore, the assay was sensitive enough to evaluate multiplex gene targeting. This method is easy to implement by laboratories with access to a capillary sequencer. Although we validated the method using CRISPR/Cas9 and zebrafish, it can be applied to other model systems and other genome targeting nucleases.
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Sistemas CRISPR-Cas , Mutação INDEL , Reação em Cadeia da Polimerase/métodos , RNA/metabolismo , Animais , Fluorescência , Peixe-Zebra/genéticaRESUMO
With the completion of the zebrafish genome sequencing project, it becomes possible to analyze the function of zebrafish genes in a systematic way. The first step in such an analysis is to inactivate each protein-coding gene by targeted or random mutation. Here we describe a streamlined pipeline using proviral insertions coupled with high-throughput sequencing and mapping technologies to widely mutagenize genes in the zebrafish genome. We also report the first 6144 mutagenized and archived F1's predicted to carry up to 3776 mutations in annotated genes. Using in vitro fertilization, we have rescued and characterized ~0.5% of the predicted mutations, showing mutation efficacy and a variety of phenotypes relevant to both developmental processes and human genetic diseases. Mutagenized fish lines are being made freely available to the public through the Zebrafish International Resource Center. These fish lines establish an important milestone for zebrafish genetics research and should greatly facilitate systematic functional studies of the vertebrate genome.
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Técnicas de Inativação de Genes , Estudo de Associação Genômica Ampla , Genômica , Peixe-Zebra/genética , Alelos , Animais , Mapeamento Cromossômico/métodos , Biologia Computacional/métodos , Gammaretrovirus/fisiologia , Anotação de Sequência Molecular , Mutagênese Insercional , Mutação , Fenótipo , Integração ViralRESUMO
UNLABELLED: There are several experimental contexts in which it is important to identify DNA integration sites, such as insertional mutagenesis screens, gene and enhancer trap applications, and gene therapy. We previously developed an assay to identify millions of integrations in multiplexed barcoded samples at base-pair resolution. The sheer amount of data produced by this approach makes the mapping of individual sites non-trivial without bioinformatics support. This article presents the Genomic Integration Site Tracker (GeIST), a command-line pipeline designed to map the integration sites produced by this assay and identify the samples from which they came. GeIST version 2.1.0, a more adaptable version of our original pipeline, can identify integrations of murine leukemia virus, adeno-associated virus, Tol2 transposons or Ac/Ds transposons, and can be adapted for other inserted elements. It has been tested on experimental data for each of these delivery vectors and fine-tuned to account for sequencing and cloning artifacts. AVAILABILITY AND IMPLEMENTATION: GeIST uses a combination of Bash shell scripting and Perl. GeIST is available at http://research.nhgri.nih.gov/software/GeIST/. CONTACT: burgess@mail.nih.gov.
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Mapeamento Cromossômico , Biologia Computacional/métodos , Elementos de DNA Transponíveis/genética , Dependovirus/genética , Genômica/métodos , Vírus da Leucemia Murina/genética , Mutagênese Insercional , Animais , Bases de Dados Genéticas , Marcadores Genéticos , Humanos , Camundongos , Fluxo de TrabalhoRESUMO
G(M2) gangliosidoses are severe neurodegenerative disorders resulting from a deficiency in ß-hexosaminidase A activity and lacking effective therapies. Using a Sandhoff disease (SD) mouse model (Hexb(-/-)) of the G(M2) gangliosidoses, we tested the potential of systemically delivered adeno-associated virus 9 (AAV9) expressing Hexb cDNA to correct the neurological phenotype. Neonatal or adult SD and normal mice were intravenously injected with AAV9-HexB or -LacZ and monitored for serum ß-hexosaminidase activity, motor function, and survival. Brain G(M2) ganglioside, ß-hexosaminidase activity, and inflammation were assessed at experimental week 43, or an earlier humane end point. SD mice injected with AAV9-LacZ died by 17 weeks of age, whereas all neonatal AAV9-HexB-treated SD mice survived until 43 weeks (P < 0.0001) with only three exhibiting neurological dysfunction. SD mice treated as adults with AAV9-HexB died between 17 and 35 weeks. Neonatal SD-HexB-treated mice had a significant increase in brain ß-hexosaminidase activity, and a reduction in G(M2) ganglioside storage and neuroinflammation compared to adult SD-HexB- and SD-LacZ-treated groups. However, at 43 weeks, 8 of 10 neonatal-HexB injected control and SD mice exhibited liver or lung tumors. This study demonstrates the potential for long-term correction of SD and other G(M2) gangliosidoses through early rAAV9 based systemic gene therapy.
Assuntos
Dependovirus/genética , Gangliosídeo G(M2)/metabolismo , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Doença de Sandhoff/terapia , Cadeia beta da beta-Hexosaminidase/genética , Fatores Etários , Animais , Animais Recém-Nascidos , Encéfalo/enzimologia , Encéfalo/patologia , Modelos Animais de Doenças , Feminino , Expressão Gênica , Vetores Genéticos/efeitos adversos , Inflamação/genética , Inflamação/mortalidade , Inflamação/patologia , Inflamação/terapia , Injeções Intravenosas , Óperon Lac , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/patologia , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/patologia , Lisossomos/enzimologia , Lisossomos/patologia , Masculino , Camundongos , Camundongos Knockout , Atividade Motora/genética , Doença de Sandhoff/genética , Doença de Sandhoff/mortalidade , Doença de Sandhoff/patologia , Análise de Sobrevida , Cadeia beta da beta-Hexosaminidase/metabolismoRESUMO
Retroviruses integrate into the host genome in patterns specific to each virus. Understanding the causes of these patterns can provide insight into viral integration mechanisms, pathology and genome evolution, and is critical to the development of safe gene therapy vectors. We generated murine leukemia virus integrations in human HepG2 and K562 cells and subjected them to second-generation sequencing, using a DNA barcoding technique that allowed us to quantify independent integration events. We characterized >3,700,000 unique integration events in two ENCODE-characterized cell lines. We find that integrations were most highly enriched in a subset of strong enhancers and active promoters. In both cell types, approximately half the integrations were found in <2% of the genome, demonstrating genomic influences even narrower than previously believed. The integration pattern of murine leukemia virus appears to be largely driven by regions that have high enrichment for multiple marks of active chromatin; the combination of histone marks present was sufficient to explain why some strong enhancers were more prone to integration than others. The approach we used is applicable to analyzing the integration pattern of any exogenous element and could be a valuable preclinical screen to evaluate the safety of gene therapy vectors.
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Sítios de Ligação Microbiológicos , Elementos Facilitadores Genéticos , Vírus da Leucemia Murina de Moloney/fisiologia , Regiões Promotoras Genéticas , Integração Viral , Linhagem Celular Tumoral , Humanos , Células K562RESUMO
ZInC (Zebrafish Insertional Collection, http://research.nhgri.nih.gov/ZInC/) is a web-searchable interface of insertional mutants in zebrafish. Over the last two decades, the zebrafish has become a popular model organism for studying vertebrate development as well as for modeling human diseases. To facilitate such studies, we are generating a genome-wide knockout resource that targets every zebrafish protein-coding gene. All mutant fish are freely available to the scientific community through the Zebrafish International Resource Center (ZIRC). To assist researchers in finding mutant and insertion information, we developed a comprehensive database with a web front-end, the ZInC. It can be queried using multiple types of input such as ZFIN (Zebrafish Information Network) IDs, UniGene accession numbers and gene symbols from zebrafish, human and mouse. In the future, ZInC may include data from other insertional mutation projects as well. ZInC cross-references all integration data with the ZFIN (http://zfin.org/).