RESUMEN
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.
Asunto(s)
Sistemas CRISPR-Cas , Marcación de Gen , Ensayos Analíticos de Alto Rendimiento , Fenotipo , Alelos , Animales , Técnicas de Inactivación de Genes , Marcación de Gen/métodos , Estudio de Asociación del Genoma Completo , Genómica , Células Germinativas/inmunología , Humanos , Mutagénesis , Sitios de Carácter Cuantitativo , ARN Guía de Kinetoplastida/genética , Eliminación de Secuencia , Pez CebraRESUMEN
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.
Asunto(s)
Mapeo Cromosómico , Biología Computacional/métodos , Elementos Transponibles de ADN/genética , Dependovirus/genética , Genómica/métodos , Virus de la Leucemia Murina/genética , Mutagénesis Insercional , Animales , Bases de Datos Genéticas , Marcadores Genéticos , Humanos , Ratones , Flujo de TrabajoRESUMEN
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.
Asunto(s)
Dependovirus/genética , Gangliósido G(M2)/metabolismo , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Enfermedad de Sandhoff/terapia , Cadena beta de beta-Hexosaminidasa/genética , Factores de Edad , Animales , Animales Recién Nacidos , Encéfalo/enzimología , Encéfalo/patología , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Vectores Genéticos/efectos adversos , Inflamación/genética , Inflamación/mortalidad , Inflamación/patología , Inflamación/terapia , Inyecciones Intravenosas , Operón Lac , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/patología , Neoplasias Pulmonares/etiología , Neoplasias Pulmonares/patología , Lisosomas/enzimología , Lisosomas/patología , Masculino , Ratones , Ratones Noqueados , Actividad Motora/genética , Enfermedad de Sandhoff/genética , Enfermedad de Sandhoff/mortalidad , Enfermedad de Sandhoff/patología , Análisis de Supervivencia , Cadena beta de beta-Hexosaminidasa/metabolismoRESUMEN
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.
Asunto(s)
Sitios de Ligazón Microbiológica , Elementos de Facilitación Genéticos , Virus de la Leucemia Murina de Moloney/fisiología , Regiones Promotoras Genéticas , Integración Viral , Línea Celular Tumoral , Humanos , Células K562RESUMEN
BACKGROUND: Quantification of a transcriptional profile is a useful way to evaluate the activity of a cell at a given point in time. Although RNA-Seq has revolutionized transcriptional profiling, the costs of RNA-Seq are still significantly higher than microarrays, and often the depth of data delivered from RNA-Seq is in excess of what is needed for simple transcript quantification. Digital Gene Expression (DGE) is a cost-effective, sequence-based approach for simple transcript quantification: by sequencing one read per molecule of RNA, this technique can be used to efficiently count transcripts while obviating the need for transcript-length normalization and reducing the total numbers of reads necessary for accurate quantification. Here, we present trieFinder, a program specifically designed to rapidly map, parse, and annotate DGE tags of various lengths against cDNA and/or genomic sequence databases. RESULTS: The trieFinder algorithm maps DGE tags in a two-step process. First, it scans FASTA files of RefSeq, UniGene, and genomic DNA sequences to create a database of all tags that can be derived from a predefined restriction site. Next, it compares the experimental DGE tags to this tag database, taking advantage of the fact that the tags are stored as a prefix tree, or "trie", which allows for linear-time searches for exact matches. DGE tags with mismatches are analyzed by recursive calls in the data structure. We find that, in terms of alignment speed, the mapping functionality of trieFinder compares favorably with Bowtie. CONCLUSIONS: trieFinder can quickly provide the user an annotation of the DGE tags from three sources simultaneously, simplifying transcript quantification and novel transcript detection, delivering the data in a simple parsed format, obviating the need to post-process the alignment results. trieFinder is available at http://research.nhgri.nih.gov/software/trieFinder/.
Asunto(s)
Algoritmos , Perfilación de la Expresión Génica/métodos , Anotación de Secuencia Molecular/métodos , ADN Complementario/genética , Bases de Datos Genéticas , Genómica , Análisis de Secuencia de ADN , Programas InformáticosRESUMEN
The MRN complex, composed of MRE11, RAD50 and NBS, plays important roles in responding to DNA double-strand breaks (DSBs). In metazoans, functional studies of genes encoding these proteins have been challenging because complete loss-of-function mutations are lethal at the organismal level and because NBS has multiple functions in DNA damage responses. To study functions of Drosophila NBS in DNA damage responses, we used a separation-of-function mutation that causes loss of the forkhead-associated (FHA) domain. Loss of the FHA domain resulted in hypersensitivity to ionizing radiation and defects in gap repair by homologous recombination, but had only a small effect on the DNA damage checkpoint response and did not impair DSB repair by end joining. We also found that heterozygosity for an nbs null mutation caused reduced gap repair and loss of the checkpoint response to low-dose irradiation. These findings shed light on possible sources of the cancer predisposition found in human carriers of NBN mutations.
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Proteínas Portadoras/fisiología , Daño del ADN , Proteínas de Drosophila/fisiología , Mutación , Animales , Animales Modificados Genéticamente , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN/fisiología , Drosophila/genética , Drosophila/metabolismo , Drosophila/efectos de la radiación , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Femenino , Rayos gamma , Larva/genética , Larva/metabolismo , Larva/efectos de la radiación , Masculino , Recombinación GenéticaRESUMEN
The zebrafish is a popular model organism for studying development and disease, and genetically modified zebrafish provide an essential tool for functional genomic studies. Numerous publications have demonstrated the efficacy of gene targeting in zebrafish using CRISPR/Cas9, and they have included descriptions of a variety of tools and methods for guide RNA synthesis and mutant identification. However, most of the published techniques are not readily scalable to increase throughput. We recently described a CRISPR/Cas9-based high-throughput mutagenesis and phenotyping pipeline in zebrafish. Here, we present a complete workflow for this pipeline, including target selection; cloning-free single-guide RNA (sgRNA) synthesis; microinjection; validation of the target-specific activity of the sgRNAs; founder screening to identify germline-transmitting mutations by fluorescence PCR; determination of the exact lesion by Sanger or next-generation sequencing (including software for analysis); and genotyping in the F1 or subsequent generations. Using these methods, sgRNAs can be evaluated in 3 d, zebrafish germline-transmitting mutations can be identified within 3 months and stable lines can be established within 6 months. Realistically, two researchers can target tens to hundreds of genes per year using this protocol.
Asunto(s)
Sistemas CRISPR-Cas/genética , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutagénesis , Pez Cebra/genética , AnimalesRESUMEN
DNA transposons and retroviruses are important transgenic tools for genome engineering. An important consideration affecting the choice of transgenic vector is their insertion site preferences. Previous large-scale analyses of Ds transposon integration sites in plants were done on the basis of reporter gene expression or germ-line transmission, making it difficult to discern vertebrate integration preferences. Here, we compare over 1300 Ds transposon integration sites in zebrafish with Tol2 transposon and retroviral integration sites. Genome-wide analysis shows that Ds integration sites in the presence or absence of marker selection are remarkably similar and distributed throughout the genome. No strict motif was found, but a preference for structural features in the target DNA associated with DNA flexibility (Twist, Tilt, Rise, Roll, Shift, and Slide) was observed. Remarkably, this feature is also found in transposon and retroviral integrations in maize and mouse cells. Our findings show that structural features influence the integration of heterologous DNA in genomes, and have implications for targeted genome engineering.
Asunto(s)
Elementos Transponibles de ADN , Estudio de Asociación del Genoma Completo , Genoma , Genómica , Retroviridae/genética , Integración Viral , Animales , Secuencia de Bases , Marcación de Gen , Ingeniería Genética , Genómica/métodos , Ratones , Virus de la Leucemia Murina de Moloney/genética , Mutagénesis Insercional , Motivos de Nucleótidos , Secuencias Repetitivas de Ácidos Nucleicos , Pez Cebra/genéticaRESUMEN
Determining the mechanism of gene function is greatly enhanced using conditional mutagenesis. However, generating engineered conditional alleles is inefficient and has only been widely used in mice. Importantly, multiplex conditional mutagenesis requires extensive breeding. Here we demonstrate a system for one-generation multiplex conditional mutagenesis in zebrafish (Danio rerio) using transgenic expression of both cas9 and multiple single guide RNAs (sgRNAs). We describe five distinct zebrafish U6 promoters for sgRNA expression and demonstrate efficient multiplex biallelic inactivation of tyrosinase and insulin receptor a and b, resulting in defects in pigmentation and glucose homeostasis. Furthermore, we demonstrate temporal and tissue-specific mutagenesis using transgenic expression of Cas9. Heat-shock-inducible expression of cas9 allows temporal control of tyr mutagenesis. Liver-specific expression of cas9 disrupts insulin receptor a and b, causing fasting hypoglycemia and postprandial hyperglycemia. We also show that delivery of sgRNAs targeting ascl1a into the eye leads to impaired damage-induced photoreceptor regeneration. Our findings suggest that CRISPR/Cas9-based conditional mutagenesis in zebrafish is not only feasible but rapid and straightforward.
Asunto(s)
Sistemas CRISPR-Cas , Expresión Génica , Marcación de Gen , Mutagénesis , ARN Guía de Kinetoplastida/genética , Transgenes , Animales , Animales Modificados Genéticamente , Orden Génico , Silenciador del Gen , Vectores Genéticos/genética , Glucosa/metabolismo , Hipopigmentación/genética , Fenotipo , Pez CebraRESUMEN
Since the sequencing of the human reference genome, many human disease-related genes have been discovered. However, understanding the functions of all the genes in the genome remains a challenge. The biological activities of these genes are usually investigated in model organisms such as mice and zebrafish. Large-scale mutagenesis screens to generate disruptive mutations are useful for identifying and understanding the activities of genes. Here, we report a multifunctional mutagenesis system in zebrafish using the maize Ds transposon. Integration of the Ds transposable element containing an mCherry reporter for protein trap events and an EGFP reporter for enhancer trap events produced a collection of transgenic lines marking distinct cell and tissue types, and mutagenized genes in the zebrafish genome by trapping and prematurely terminating endogenous protein coding sequences. We obtained 642 zebrafish lines with dynamic reporter gene expression. The characterized fish lines with specific expression patterns will be made available through the European Zebrafish Resource Center (EZRC), and a database of reporter expression is available online (http://fishtrap.warwick.ac.uk/). Our approach complements other efforts using zebrafish to facilitate functional genomic studies in this model of human development and disease.
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Mutagénesis Insercional/métodos , Pez Cebra/genética , Animales , Secuencia de Bases , Mapeo Cromosómico , Elementos de Facilitación Genéticos , Fluorescencia , Perfilación de la Expresión Génica , Genes Reporteros , Sitios Genéticos , Datos de Secuencia Molecular , Mutación/genética , Especificidad de Órganos/genética , Fenotipo , Proteínas de Pez Cebra/genéticaRESUMEN
Transgenic methods enable the selective manipulation of neurons for functional mapping of neuronal circuits. Using confocal microscopy, we have imaged the cellular-level expression of 109 transgenic lines in live 6 day post fertilization larvae, including 80 Gal4 enhancer trap lines, 9 Cre enhancer trap lines and 20 transgenic lines that express fluorescent proteins in defined gene-specific patterns. Image stacks were acquired at single micron resolution, together with a broadly expressed neural marker, which we used to align enhancer trap reporter patterns into a common 3-dimensional reference space. To facilitate use of this resource, we have written software that enables searching for transgenic lines that label cells within a selectable 3-dimensional region of interest (ROI) or neuroanatomical area. This software also enables the intersectional expression of transgenes to be predicted, a feature which we validated by detecting cells with co-expression of Cre and Gal4. Many of the imaged enhancer trap lines show intrinsic brain-specific expression. However, to increase the utility of lines that also drive expression in non-neuronal tissue we have designed a novel UAS reporter, that suppresses expression in heart, muscle, and skin through the incorporation of microRNA binding sites in a synthetic 3' untranslated region. Finally, we mapped the site of transgene integration, thus providing molecular identification of the expression pattern for most lines. Cumulatively, this library of enhancer trap lines provides genetic access to 70% of the larval brain and is therefore a powerful and broadly accessible tool for the dissection of neural circuits in larval zebrafish.
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Bases de Datos Factuales , Imagenología Tridimensional/métodos , Pez Cebra/anatomía & histología , Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente/anatomía & histología , Animales Modificados Genéticamente/crecimiento & desarrollo , Animales Modificados Genéticamente/metabolismo , Encéfalo/citología , Encéfalo/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Expresión Génica , MicroARNs/genética , MicroARNs/metabolismo , Microscopía Confocal/métodos , Músculos/citología , Músculos/metabolismo , Miocardio/citología , Miocardio/metabolismo , Piel/citología , Piel/metabolismo , Programas Informáticos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The use of adeno-associated virus (AAV) as a gene therapy vector has been approved recently for clinical use and has demonstrated efficacy in a growing number of clinical trials. However, the safety of AAV as a vector has been challenged by a single study that documented hepatocellular carcinoma (HCC) after AAV gene delivery in mice. Most studies have not noted genotoxicity following AAV-mediated gene delivery; therefore, the possibility that there is an association between AAV and HCC is controversial. Here, we performed a comprehensive study of HCC in a large number of mice following therapeutic AAV gene delivery. Using a sensitive high-throughput integration site-capture technique and global expressional analysis, we found that AAV integration into the RNA imprinted and accumulated in nucleus (Rian) locus, and the resulting overexpression of proximal microRNAs and retrotransposon-like 1 (Rtl1) were associated with HCC. In addition, we demonstrated that the AAV vector dose, enhancer/promoter selection, and the timing of gene delivery are all critical factors for determining HCC incidence after AAV gene delivery. Together, our results define aspects of AAV-mediated gene therapy that influence genotoxicity and suggest that these features should be considered for design of both safer AAV vectors and gene therapy studies.
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Carcinoma Hepatocelular , Dependovirus , Terapia Genética/métodos , Vectores Genéticos , Neoplasias Hepáticas , Transducción Genética , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/terapia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/terapia , Ratones , Ratones Mutantes , Proteínas Gestacionales/genética , Proteínas Gestacionales/metabolismoRESUMEN
Genetic screens are a powerful tool to discover genes that are important in immune cell development and function. The evolutionarily conserved development of lymphoid cells paired with the genetic tractability of zebrafish make this a powerful model system for this purpose. We used a Tol2-based gene-breaking transposon to induce mutations in the zebrafish (Danio rerio, AB strain) genome, which served the dual purpose of fluorescently tagging cells and tissues that express the disrupted gene and provided a means of identifying the disrupted gene. We identified 12 lines in which hematopoietic tissues expressed green fluorescent protein (GFP) during embryonic development, as detected by microscopy. Subsequent analysis of young adult fish, using a novel approach in which single cell suspensions of whole fish were analyzed by flow cytometry, revealed that 8 of these lines also exhibited GFP expression in young adult cells. An additional 15 lines that did not have embryonic GFP+ hematopoietic tissue by microscopy, nevertheless exhibited GFP+ cells in young adults. RT-PCR analysis of purified GFP+ populations for expression of T and B cell-specific markers identified 18 lines in which T and/or B cells were fluorescently tagged at 6 weeks of age. As transposon insertion is expected to cause gene disruption, these lines can be used to assess the requirement for the disrupted genes in immune cell development. Focusing on the lines with embryonic GFP+ hematopoietic tissue, we identified three lines in which homozygous mutants exhibited impaired T cell development at 6 days of age. In two of the lines we identified the disrupted genes, agtpbp1 and eps15L1. Morpholino-mediated knockdown of these genes mimicked the T cell defects in the corresponding mutant embryos, demonstrating the previously unrecognized, essential roles of agtpbp1 and eps15L1 in T cell development.
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Carboxipeptidasas/genética , Linfocitos T/fisiología , Proteínas de Pez Cebra/genética , Animales , Carboxipeptidasas/metabolismo , Diferenciación Celular , Expresión Génica , Técnicas de Silenciamiento del Gen , Hematopoyesis , Mutagénesis , Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
UNLABELLED: Pseudomonas aeruginosa is an antibiotic-refractory pathogen with a large genome and extensive genotypic diversity. Historically, P. aeruginosa has been a major model system for understanding the molecular mechanisms underlying type I clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR-Cas)-based bacterial immune system function. However, little information on the phylogenetic distribution and potential role of these CRISPR-Cas systems in molding the P. aeruginosa accessory genome and antibiotic resistance elements is known. Computational approaches were used to identify and characterize CRISPR-Cas systems within 672 genomes, and in the process, we identified a previously unreported and putatively mobile type I-C P. aeruginosa CRISPR-Cas system. Furthermore, genomes harboring noninhibited type I-F and I-E CRISPR-Cas systems were on average ~300 kb smaller than those without a CRISPR-Cas system. In silico analysis demonstrated that the accessory genome (n = 22,036 genes) harbored the majority of identified CRISPR-Cas targets. We also assembled a global spacer library that aided the identification of difficult-to-characterize mobile genetic elements within next-generation sequencing (NGS) data and allowed CRISPR typing of a majority of P. aeruginosa strains. In summary, our analysis demonstrated that CRISPR-Cas systems play an important role in shaping the accessory genomes of globally distributed P. aeruginosa isolates. IMPORTANCE: P. aeruginosa is both an antibiotic-refractory pathogen and an important model system for type I CRISPR-Cas bacterial immune systems. By combining the genome sequences of 672 newly and previously sequenced genomes, we were able to provide a global view of the phylogenetic distribution, conservation, and potential targets of these systems. This analysis identified a new and putatively mobile P. aeruginosa CRISPR-Cas subtype, characterized the diverse distribution of known CRISPR-inhibiting genes, and provided a potential new use for CRISPR spacer libraries in accessory genome analysis. Our data demonstrated the importance of CRISPR-Cas systems in modulating the accessory genomes of globally distributed strains while also providing substantial data for subsequent genomic and experimental studies in multiple fields. Understanding why certain genotypes of P. aeruginosa are clinically prevalent and adept at horizontally acquiring virulence and antibiotic resistance elements is of major clinical and economic importance.
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Antibacterianos/farmacología , Sistemas CRISPR-Cas , Farmacorresistencia Bacteriana , Variación Genética , Filogenia , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Biología Computacional , Genoma Bacteriano , Pseudomonas aeruginosa/clasificación , Análisis de Secuencia de ADNRESUMEN
Substantial intrastrain variation at the nucleotide level complicates molecular and genetic studies in zebrafish, such as the use of CRISPRs or morpholinos to inactivate genes. In the absence of robust inbred zebrafish lines, we generated NHGRI-1, a healthy and fecund strain derived from founder parents we sequenced to a depth of â¼50×. Within this strain, we have identified the majority of the genome that matches the reference sequence and documented most of the variants. This strain has utility for many reasons, but in particular it will be useful for any researcher who needs to know the exact sequence (with all variants) of a particular genomic region or who wants to be able to robustly map sequences back to a genome with all possible variants defined.
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Genómica/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Pez Cebra/genética , Animales , Animales EndogámicosRESUMEN
The Bloom syndrome helicase, BLM, has numerous functions that prevent mitotic crossovers. We used unique features of Drosophila melanogaster to investigate origins and properties of mitotic crossovers that occur when BLM is absent. Induction of lesions that block replication forks increased crossover frequencies, consistent with functions for BLM in responding to fork blockage. In contrast, treatment with hydroxyurea, which stalls forks, did not elevate crossovers, even though mutants lacking BLM are sensitive to killing by this agent. To learn about sources of spontaneous recombination, we mapped mitotic crossovers in mutants lacking BLM. In the male germline, irradiation-induced crossovers were distributed randomly across the euchromatin, but spontaneous crossovers were nonrandom. We suggest that regions of the genome with a high frequency of mitotic crossovers may be analogous to common fragile sites in the human genome. Interestingly, in the male germline there is a paucity of crossovers in the interval that spans the pericentric heterochromatin, but in the female germline this interval is more prone to crossing over. Finally, our system allowed us to recover pairs of reciprocal crossover chromosomes. Sequencing of these revealed the existence of gene conversion tracts and did not provide any evidence for mutations associated with crossovers. These findings provide important new insights into sources and structures of mitotic crossovers and functions of BLM helicase.
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Intercambio Genético/genética , Reparación del ADN por Unión de Extremidades/genética , ADN Helicasas/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Animales , Secuencia de Bases , Intercambio Genético/efectos de los fármacos , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Femenino , Hidroxiurea/farmacología , Masculino , Mitosis/genética , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Análisis de Secuencia de ADNRESUMEN
Numerous transposable element insertions in Drosophila melanogaster cause hypomorphic mutations. We report that transcription initiation within a region found in many P-element constructs provides an explanation for why some gene function is retained. We detected evidence of this transcription in four different types of P constructs, regardless of whether the insertion was in a coding exon, intron, 5' untranslated region, or upstream of the gene span.
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Elementos Transponibles de ADN , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Genes de Insecto , Larva/genética , Pupa/genética , ARN Mensajero , Transcripción Genética , Regiones no Traducidas 5' , Alelos , Animales , Secuencia de Bases , Exones , Femenino , Expresión Génica , Intrones , Masculino , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutación , Fenotipo , ARN Mensajero/análisis , ARN Mensajero/química , ARN Mensajero/genéticaRESUMEN
Two commonly used promoters to ubiquitously express transgenes in zebrafish are the Xenopus laevis elongation factor 1 alpha promoter (XlEef1a1) and the zebrafish histone variant H2A.F/Z (h2afv) promoter. Recently, transgenes utilizing these promoters were shown to be silenced in certain adult tissues, particularly the central nervous system. To overcome this limitation, we cloned the promoters of four zebrafish genes that likely are transcribed ubiquitously throughout development and into the adult. These four genes are the TATA box binding protein gene, the taube nuss-like gene, the eukaryotic elongation factor 1-gamma gene, and the beta-actin-1 gene. We PCR amplified approximately 2.5 kb upstream of the putative translational start site of each gene and cloned each into a Tol2 expression vector that contains the EGFP reporter transgene. We used these four Tol2 vectors to independently generate stable transgenic fish lines for analysis of transgene expression during development and in the adult. We demonstrated that all four promoters drive a very broad pattern of EGFP expression throughout development and the adult. Using the retina as a well-characterized component of the CNS, all four promoters appeared to drive EGFP expression in all neuronal and non-neuronal cells of the adult retina. In contrast, the h2afv promoter failed to express EGFP in the adult retina. When we examined EGFP expression in the various cells of the blood cell lineage, we observed that all four promoters exhibited a more heterogenous expression pattern than either the XlEef1a1 or h2afv promoters. While these four ubiquitous promoters did not express EGFP in all the adult blood cells, they did express EGFP throughout the CNS and in broader expression patterns in the adult than either the XlEef1a1 or h2afv promoters. For these reasons, these four promoters will be valuable tools for expressing transgenes in adult zebrafish.