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1.
Sci Adv ; 5(6): eaav0547, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31249862

RESUMO

For over a thousand years, the common goldfish (Carassius auratus) was raised throughout Asia for food and as an ornamental pet. As a very close relative of the common carp (Cyprinus carpio), goldfish share the recent genome duplication that occurred approximately 14 million years ago in their common ancestor. The combination of centuries of breeding and a wide array of interesting body morphologies provides an exciting opportunity to link genotype to phenotype and to understand the dynamics of genome evolution and speciation. We generated a high-quality draft sequence and gene annotations of a "Wakin" goldfish using 71X PacBio long reads. The two subgenomes in goldfish retained extensive synteny and collinearity between goldfish and zebrafish. However, genes were lost quickly after the carp whole-genome duplication, and the expression of 30% of the retained duplicated gene diverged substantially across seven tissues sampled. Loss of sequence identity and/or exons determined the divergence of the expression levels across all tissues, while loss of conserved noncoding elements determined expression variance between different tissues. This assembly provides an important resource for comparative genomics and understanding the causes of goldfish variants.

2.
Methods Mol Biol ; 1874: 459-474, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30353530

RESUMO

A major strength of zebrafish as a model organism is their rapid, in vitro development. The easy access to embryos compared to mammals, allows larval molecular and cellular composition to be manipulated by microinjection, providing a powerful avenue for biological and translational studies. Here, we describe the essential steps and different applications of microinjection in zebrafish for genome editing and functional studies, along with some experimental tips that are critical for microinjection success.


Assuntos
Edição de Genes/métodos , Microinjeções/métodos , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados/crescimento & desenvolvimento , Sistemas CRISPR-Cas , Chaperonina 60/genética , Peixe-Zebra/genética
3.
Front Genet ; 9: 348, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30233640

RESUMO

Hematopoiesis results in the correct formation of all the different blood cell types. In mammals, it starts from specific hematopoietic stem and precursor cells residing in the bone marrow. Mature blood cells are responsible for supplying oxygen to every cell of the organism and for the protection against pathogens. Therefore, inherited or de novo genetic mutations affecting blood cell formation or the regulation of their activity are responsible for numerous diseases including anemia, immunodeficiency, autoimmunity, hyper- or hypo-inflammation, and cancer. By definition, an animal disease model is an analogous version of a specific clinical condition developed by researchers to gain information about its pathophysiology. Among all the model species used in comparative medicine, mice continue to be the most common and accepted model for biomedical research. However, because of the complexity of human diseases and the intrinsic differences between humans and other species, the use of several models (possibly in distinct species) can often be more helpful and informative than the use of a single model. In recent decades, the zebrafish (Danio rerio) has become increasingly popular among researchers, because it represents an inexpensive alternative compared to mammalian models, such as mice. Numerous advantages make it an excellent animal model to be used in genetic studies and in particular in modeling human blood diseases. Comparing zebrafish hematopoiesis to mammals, it is highly conserved with few, significant differences. In addition, the zebrafish model has a high-quality, complete genomic sequence available that shows a high level of evolutionary conservation with the human genome, empowering genetic and genomic approaches. Moreover, the external fertilization, the high fecundity and the transparency of their embryos facilitate rapid, in vivo analysis of phenotypes. In addition, the ability to manipulate its genome using the last genome editing technologies, provides powerful tools for developing new disease models and understanding the pathophysiology of human disorders. This review provides an overview of the different approaches and techniques that can be used to model genetic diseases in zebrafish, discussing how this animal model has contributed to the understanding of genetic diseases, with a specific focus on the blood disorders.

4.
Front Cell Dev Biol ; 6: 88, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30186835

RESUMO

Thousands of genes have been implicated in retinal regeneration, but only a few have been shown to impact the regenerative capacity of Müller glia-an adult retinal stem cell with untapped therapeutic potential. Similarly, among nearly 300 genetic loci associated with human retinal disease, the majority remain untested in animal models. To address the large-scale nature of these problems, we are applying CRISPR/Cas9-based genome modification strategies in zebrafish to target over 300 genes implicated in retinal regeneration or degeneration. Our intent is to enable large-scale reverse genetic screens by applying a multiplexed gene disruption strategy that markedly increases the efficiency of the screening process. To facilitate large-scale phenotyping, we incorporate an automated reporter quantification-based assay to identify cellular degeneration and regeneration-deficient phenotypes in transgenic fish. Multiplexed gene targeting strategies can address mismatches in scale between "big data" bioinformatics and wet lab experimental capacities, a critical shortfall limiting comprehensive functional analyses of factors implicated in ever-expanding multiomics datasets. This report details the progress we have made to date with a multiplexed CRISPR/Cas9-based gene targeting strategy and discusses how the methodologies applied can further our understanding of the genes that predispose to retinal degenerative disease and which control the regenerative capacity of retinal Müller glia cells.

5.
Dis Model Mech ; 11(9)2018 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-30135069

RESUMO

Niemann-Pick disease type C1 (NPC1) is a rare autosomal recessive lysosomal storage disease primarily caused by mutations in NPC1 NPC1 is characterized by abnormal accumulation of unesterified cholesterol and glycolipids in late endosomes and lysosomes. Common signs include neonatal jaundice, hepatosplenomegaly, cerebellar ataxia, seizures and cognitive decline. Both mouse and feline models of NPC1 mimic the disease progression in humans and have been used in preclinical studies of 2-hydroxypropyl-ß-cyclodextrin (2HPßCD; VTS-270), a drug that appeared to slow neurological progression in a Phase 1/2 clinical trial. However, there remains a need to identify additional therapeutic agents. High-throughput drug screens have been useful in identifying potential therapeutic compounds; however, current preclinical testing is time and labor intensive. Thus, development of a high-capacity in vivo platform suitable for screening candidate drugs/compounds would be valuable for compound optimization and prioritizing subsequent in vivo testing. Here, we generated and characterize two zebrafish npc1-null mutants using CRISPR/Cas9-mediated gene targeting. The npc1 mutants model both the early liver and later neurological disease phenotypes of NPC1. LysoTracker staining of npc1 mutant larvae was notable for intense staining of lateral line neuromasts, thus providing a robust in vivo screen for lysosomal storage. As a proof of principle, we were able to show that treatment of the npc1 mutant larvae with 2HPßCD significantly reduced neuromast LysoTracker staining. These data demonstrate the potential value of using this zebrafish NPC1 model for efficient and rapid in vivo optimization and screening of potential therapeutic compounds.This article has an associated First Person interview with the first author of the paper.

6.
NPJ Regen Med ; 3: 11, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29872546

RESUMO

Regenerative medicine holds great promise for both degenerative diseases and traumatic tissue injury which represent significant challenges to the health care system. Hearing loss, which affects hundreds of millions of people worldwide, is caused primarily by a permanent loss of the mechanosensory receptors of the inner ear known as hair cells. This failure to regenerate hair cells after loss is limited to mammals, while all other non-mammalian vertebrates tested were able to completely regenerate these mechanosensory receptors after injury. To understand the mechanism of hair cell regeneration and its association with regeneration of other tissues, we performed a guided mutagenesis screen using zebrafish lateral line hair cells as a screening platform to identify genes that are essential for hair cell regeneration, and further investigated how genes essential for hair cell regeneration were involved in the regeneration of other tissues. We created genetic mutations either by retroviral insertion or CRISPR/Cas9 approaches, and developed a high-throughput screening pipeline for analyzing hair cell development and regeneration. We screened 254 gene mutations and identified 7 genes specifically affecting hair cell regeneration. These hair cell regeneration genes fell into distinct and somewhat surprising functional categories. By examining the regeneration of caudal fin and liver, we found these hair cell regeneration genes often also affected other types of tissue regeneration. Therefore, our results demonstrate guided screening is an effective approach to discover regeneration candidates, and hair cell regeneration is associated with other tissue regeneration.

7.
Ann Rheum Dis ; 77(4): 612-619, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29358286

RESUMO

OBJECTIVES: To characterise the clinical features, immune manifestations and molecular mechanisms in a recently described autoinflammatory disease caused by mutations in TRNT1, a tRNA processing enzyme, and to explore the use of cytokine inhibitors in suppressing the inflammatory phenotype. METHODS: We studied nine patients with biallelic mutations in TRNT1 and the syndrome of congenital sideroblastic anaemia with immunodeficiency, fevers and developmental delay (SIFD). Genetic studies included whole exome sequencing (WES) and candidate gene screening. Patients' primary cells were used for deep RNA and tRNA sequencing, cytokine profiling, immunophenotyping, immunoblotting and electron microscopy (EM). RESULTS: We identified eight mutations in these nine patients, three of which have not been previously associated with SIFD. Three patients died in early childhood. Inflammatory cytokines, mainly interleukin (IL)-6, interferon gamma (IFN-γ) and IFN-induced cytokines were elevated in the serum, whereas tumour necrosis factor (TNF) and IL-1ß were present in tissue biopsies of patients with active inflammatory disease. Deep tRNA sequencing of patients' fibroblasts showed significant deficiency of mature cytosolic tRNAs. EM of bone marrow and skin biopsy samples revealed striking abnormalities across all cell types and a mix of necrotic and normal-appearing cells. By immunoprecipitation, we found evidence for dysregulation in protein clearance pathways. In 4/4 patients, treatment with a TNF inhibitor suppressed inflammation, reduced the need for blood transfusions and improved growth. CONCLUSIONS: Mutations of TRNT1 lead to a severe and often fatal syndrome, linking protein homeostasis and autoinflammation. Molecular diagnosis in early life will be crucial for initiating anti-TNF therapy, which might prevent some of the severe disease consequences.

8.
Hum Mol Genet ; 26(14): 2701-2718, 2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28449103

RESUMO

Mucolipidosis type IV (MLIV) is a lysosomal storage disease characterized by neurologic and ophthalmologic abnormalities. There is currently no effective treatment. MLIV is caused by mutations in MCOLN1, a lysosomal cation channel from the transient receptor potential (TRP) family. In this study, we used genome editing to knockout the two mcoln1 genes present in Danio rerio (zebrafish). Our model successfully reproduced the retinal and neuromuscular defects observed in MLIV patients, indicating that this model is suitable for studying the disease pathogenesis. Importantly, our model revealed novel insights into the origins and progression of the MLIV pathology, including the contribution of autophagosome accumulation to muscle dystrophy and the role of mcoln1 in embryonic development, hair cell viability and cellular maintenance. The generation of a MLIV model in zebrafish is particularly relevant given the suitability of this organism for large-scale in vivo drug screening, thus providing unprecedented opportunities for therapeutic discovery.


Assuntos
Mucolipidoses/genética , Canais de Receptores Transientes de Potencial/genética , Proteínas de Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Autofagossomos/metabolismo , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Mucolipidoses/metabolismo , Mucolipidoses/patologia , Mutação , Canais de Receptores Transientes de Potencial/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
9.
G3 (Bethesda) ; 7(2): 719-722, 2017 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-28040780

RESUMO

Cpf1 has emerged as an alternative to the Cas9 RNA-guided nuclease. Here we show that gene targeting rates in mice using Cpf1 can meet, or even surpass, Cas9 targeting rates (approaching 100% targeting), but require higher concentrations of mRNA and guide. We also demonstrate that coinjecting two guides with close targeting sites can result in synergistic genomic cutting, even if one of the guides has minimal cutting activity.


Assuntos
Proteínas de Bactérias/genética , Endonucleases/genética , Edição de Genes/métodos , Marcação de Genes/métodos , RNA Guia/genética , Acidaminococcus/enzimologia , Acidaminococcus/genética , Animais , Sistemas CRISPR-Cas/genética , Camundongos , RNA Mensageiro/genética
10.
Nat Protoc ; 11(12): 2357-2375, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27809318

RESUMO

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.


Assuntos
Sistemas CRISPR-Cas/genética , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutagênese , Peixe-Zebra/genética , Animais
11.
Cell Regen (Lond) ; 5: 3, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27795824

RESUMO

BACKGROUND: We are using genetics to identify genes specifically involved in hearing regeneration. In a large-scale genetic screening, we identified mgat5a, a gene in the N-glycosylation biosynthesis pathway whose activity negatively impacts hair cell regeneration. METHODS: We used a combination of mutant analysis in zebrafish and a hair cell regeneration assay to phenotype the loss of Mgat5a activity in zebrafish. We used pharmacological inhibition of N-glycosylation by swansonine. We also used over-expression analysis by mRNA injections to demonstrate how changes in N-glycosylation can alter cell signaling. RESULTS: We found that mgat5a was expressed in multiple tissues during zebrafish embryo development, particularly enriched in neural tissues including the brain, retina, and lateral line neuromasts. An mgat5a insertional mutation and a CRISPR/Cas9-generated truncation mutation both caused an enhancement of hair cell regeneration which could be phenocopied by pharmacological inhibition with swansonine. In addition to hair cell regeneration, inhibition of the N-glycosylation pathway also enhanced the regeneration of lateral line axon and caudal fins. Further analysis showed that N-glycosylation altered the responsiveness of TGF-beta signaling. CONCLUSIONS: The findings from this study provide experimental evidence for the involvement of N-glycosylation in tissue regeneration and cell signaling.

12.
Genome Biol ; 17(1): 187, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27640875

RESUMO

The search for novel ways to target and alter the genomes of living organisms accelerated rapidly this decade with the discovery of CRISPR/Cas9. Since the initial discovery, efforts to find alternative methods for altering the genome have expanded. A new study presenting an alternative approach has been demonstrated that utilizes flap endonuclease 1 (FEN-1) fused to the Fok1 endonuclease, which shows potential for DNA-guided genome targeting in vivo.


Assuntos
Endonucleases/metabolismo , Edição de Genes , Genoma , Animais , Sistemas CRISPR-Cas , DNA/química , DNA/genética , DNA/metabolismo , Endonucleases/química , Endonucleases/genética , RNA Guia/genética
13.
Sci Rep ; 6: 29946, 2016 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-27425195

RESUMO

Phosphoribosyl pyrophosphate synthetase-1 (PRPS1) is a key enzyme in nucleotide biosynthesis, and mutations in PRPS1 are found in several human diseases including nonsyndromic sensorineural deafness, Charcot-Marie-Tooth disease-5, and Arts Syndrome. We utilized zebrafish as a model to confirm that mutations in PRPS1 result in phenotypic deficiencies in zebrafish similar to those in the associated human diseases. We found two paralogs in zebrafish, prps1a and prps1b and characterized each paralogous mutant individually as well as the double mutant fish. Zebrafish prps1a mutants and prps1a;prps1b double mutants showed similar morphological phenotypes with increasingly severe phenotypes as the number of mutant alleles increased. Phenotypes included smaller eyes and reduced hair cell numbers, consistent with the optic atrophy and hearing impairment observed in human patients. The double mutant also showed abnormal development of primary motor neurons, hair cell innervation, and reduced leukocytes, consistent with the neuropathy and recurrent infection of the human patients possessing the most severe reductions of PRPS1 activity. Further analyses indicated the phenotypes were associated with a prolonged cell cycle likely resulting from reduced nucleotide synthesis and energy production in the mutant embryos. We further demonstrated the phenotypes were caused by delays in the tissues most highly expressing the prps1 genes.


Assuntos
Ribose-Fosfato Pirofosfoquinase/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Trifosfato de Adenosina/biossíntese , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Orelha Interna/embriologia , Orelha Interna/inervação , Orelha Interna/metabolismo , Embrião não Mamífero/metabolismo , Olho/metabolismo , Olho/patologia , Regulação da Expressão Gênica no Desenvolvimento , Hematopoese , Humanos , Leucócitos/metabolismo , Modelos Biológicos , Neurônios Motores/metabolismo , Mutação/genética , Fenótipo , Pigmentação/genética , Ribose-Fosfato Pirofosfoquinase/genética , S-Adenosilmetionina/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
14.
ALTEX ; 33(4): 435-452, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27328013

RESUMO

Small freshwater fish models, especially zebrafish, offer advantages over traditional rodent models, including low maintenance and husbandry costs, high fecundity, genetic diversity, physiology similar to that of traditional biomedical models, and reduced animal welfare concerns. The Collaborative Workshop on Aquatic Models and 21st Century Toxicology was held at North Carolina State University on May 5-6, 2014, in Raleigh, North Carolina, USA. Participants discussed the ways in which small fish are being used as models to screen toxicants and understand mechanisms of toxicity. Workshop participants agreed that the lack of standardized protocols is an impediment to broader acceptance of these models, whereas development of standardized protocols, validation, and subsequent regulatory acceptance would facilitate greater usage. Given the advantages and increasing application of small fish models, there was widespread interest in follow-up workshops to review and discuss developments in their use. In this article, we summarize the recommendations formulated by workshop participants to enhance the utility of small fish species in toxicology studies, as well as many of the advances in the field of toxicology that resulted from using small fish species, including advances in developmental toxicology, cardiovascular toxicology, neurotoxicology, and immunotoxicology. We alsoreview many emerging issues that will benefit from using small fish species, especially zebrafish, and new technologies that will enable using these organisms to yield results unprecedented in their information content to better understand how toxicants affect development and health.


Assuntos
Experimentação Animal , Peixes , Substâncias Perigosas/toxicidade , Testes de Toxicidade/métodos , Animais , Animais Geneticamente Modificados , Doenças Cardiovasculares/induzido quimicamente , Genoma , Genômica , Humanos , Imagem Corporal Total
16.
G3 (Bethesda) ; 6(4): 805-17, 2016 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-26818075

RESUMO

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.


Assuntos
Elementos de DNA Transponíveis , Estudo de Associação Genômica Ampla , Genoma , Genômica , Retroviridae/genética , Integração Viral , Animais , Sequência de Bases , Marcação de Genes , Engenharia Genética , Genômica/métodos , Camundongos , Vírus da Leucemia Murina de Moloney/genética , Mutagênese Insercional , Motivos de Nucleotídeos , Sequências Repetitivas de Ácido Nucleico , Peixe-Zebra/genética
17.
Nucleic Acids Res ; 44(D1): D822-6, 2016 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-26438539

RESUMO

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.


Assuntos
Sistemas CRISPR-Cas , Bases de Dados Genéticas , RNA , Peixe-Zebra/genética , Animais , Marcação de Genes , Humanos , Camundongos , Mutagênese , Peixe-Zebra/embriologia
18.
Artigo em Inglês | MEDLINE | ID: mdl-28936359

RESUMO

After injury, zebrafish can restore many tissues that do not regenerate well in mammals, making it a useful vertebrate model for studying regenerative biology. We performed a systematic screen to identify genes essential for hair cell regeneration in zebrafish, and found that the heat shock protein Hspd1 (Hsp60) has a critical role in the regeneration of hair cells and amputated caudal fins. We showed HSP60-injected extracellularly promoted cell proliferation and regeneration in both hair cells and caudal fins. We showed that hspd1 mutant was deficient in leukocyte infiltration at the site of injury. Topical application of HSP60 in a diabetic mouse skin wound model dramatically accelerated wound healing compared with controls. Stimulation of human peripheral blood mononuclear cells with HSP60 triggered a specific induction of M2 phase CD163-positive monocytes. Our results demonstrate that the normally intracellular chaperonin HSP60 has an extracellular signalling function in injury inflammation and tissue regeneration, likely through promoting the M2 phase for macrophages.

19.
Adv Genet ; 92: 1-52, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26639914

RESUMO

In the last two decades, zebrafish has become one of the fastest growing model organisms in terms of publications, however it has been plagued with the absence of a key tool in the genetics toolbox: the ability to systematically make targeted mutations in the genome. That all changed with the recent emergence of custom-built, sequence-specific nucleases, i.e., zinc finger nucleases (ZFNs), TAL-effector nucleases (TALENs), or clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas9. Here, we provide a comprehensive review of the application of these genome-editing tools to generate targeted knockout and knock-in mutants in zebrafish. These technologies have allowed us to transition from targeted knockouts in zebrafish being a difficult, resource intensive undertaking, to something that can be done in virtually any lab with modest molecular biology experience. Furthermore, this review provides a comprehensive listing of genetic and genomic resources and online tools that zebrafish researchers can use to help find a desired mutation or design effective ZFNs, TALENs, or CRISPR guide RNAs for their targeting experiments.


Assuntos
Marcação de Genes/métodos , Engenharia Genética/métodos , Peixe-Zebra/genética , Animais , Sistemas CRISPR-Cas , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Genoma , Modelos Animais
20.
Artigo em Inglês | MEDLINE | ID: mdl-26635538

RESUMO

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.


Assuntos
Bases de Dados Factuais , Imagem Tridimensional/métodos , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados/anatomia & histologia , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/metabolismo , Encéfalo/citologia , Encéfalo/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , MicroRNAs/genética , MicroRNAs/metabolismo , Microscopia Confocal/métodos , Músculos/citologia , Músculos/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Pele/citologia , Pele/metabolismo , Software , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
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