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CRISPR AND Zebrafish

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https://www.readbyqxmd.com/read/27899582/the-zebrafish-model-organism-database-new-support-for-human-disease-models-mutation-details-gene-expression-phenotypes-and-searching
#1
Douglas G Howe, Yvonne M Bradford, Anne Eagle, David Fashena, Ken Frazer, Patrick Kalita, Prita Mani, Ryan Martin, Sierra Taylor Moxon, Holly Paddock, Christian Pich, Sridhar Ramachandran, Leyla Ruzicka, Kevin Schaper, Xiang Shao, Amy Singer, Sabrina Toro, Ceri Van Slyke, Monte Westerfield
The Zebrafish Model Organism Database (ZFIN; http://zfin.org) is the central resource for zebrafish (Danio rerio) genetic, genomic, phenotypic and developmental data. ZFIN curators provide expert manual curation and integration of comprehensive data involving zebrafish genes, mutants, transgenic constructs and lines, phenotypes, genotypes, gene expressions, morpholinos, TALENs, CRISPRs, antibodies, anatomical structures, models of human disease and publications. We integrate curated, directly submitted, and collaboratively generated data, making these available to zebrafish research community...
November 28, 2016: Nucleic Acids Research
https://www.readbyqxmd.com/read/27898262/characterization-of-genetic-loss-of-function-of-fus-in-zebrafish
#2
Svetlana Lebedeva, António M de Jesus Domingues, Falk Butter, René F Ketting
The RNA-binding protein FUS is implicated in transcription, alternative splicing of neuronal genes and DNA repair. Mutations in FUS have been linked to human neurodegenerative diseases such as ALS (amyotrophic lateral sclerosis). We genetically disrupted fus in zebrafish (Danio rerio) using the CRISPR-Cas9 system. The fus knockout animals are fertile and did not show any distinctive phenotype. Mutation of fus induces mild changes in gene expression on the transcriptome and proteome level in the adult brain...
November 29, 2016: RNA Biology
https://www.readbyqxmd.com/read/27895053/utilising-polymorphisms-to-achieve-allele-specific-genome-editing-in-zebrafish
#3
Samuel J Capon, Gregory J Baillie, Neil I Bower, Jason A da Silva, Scott Paterson, Benjamin M Hogan, Cas Simons, Kelly A Smith
The advent of genome editing has significantly altered genetic research, including research using the zebrafish model. To better understand the selectivity of the commonly used CRISPR/Cas9 system, we investigated single base pair mismatches in target sites and examined how they affect genome editing in the zebrafish model. Using two different zebrafish strains that have been deep sequenced, CRISPR/Cas9 target sites containing polymorphisms between the two strains were identified. These strains were crossed (creating heterozygotes at polymorphic sites) and CRISPR/Cas9 complexes that perfectly complement one strain injected...
November 28, 2016: Biology Open
https://www.readbyqxmd.com/read/27876832/knockout-of-zebrafish-ovarian-aromatase-gene-cyp19a1a-by-talen-and-crispr-cas9-leads-to-all-male-offspring-due-to-failed-ovarian-differentiation
#4
Esther Shuk-Wa Lau, Zhiwei Zhang, Mingming Qin, Wei Ge
Sexual or gonadal differentiation is a complex event and its mechanism remains elusive in teleosts. Despite its complexity and plasticity, the process of ovarian differentiation is believed to involve gonadal aromatase (cyp19a1a) in nearly all species studied. However, most data concerning the role of aromatase have come from gene expression analysis or studies involving pharmacological approaches. There has been a lack of genetic evidence for the importance of aromatase in gonadal differentiation, especially the timing when the enzyme starts to exert its effect...
November 23, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27875093/autolysosome-biogenesis-and-developmental-senescence-are-regulated-by-both-spns1-and-v-atpase
#5
Tomoyuki Sasaki, Shanshan Lian, Alam Khan, Jesse R Llop, Andrew V Samuelson, Wenbiao Chen, Daniel J Klionsky, Shuji Kishi
Spns1 (Spinster homolog 1 [Drosophila]) in vertebrates, as well as Spin (Spinster) in Drosophila, is a hypothetical lysosomal H(+)-carbohydrate transporter, which functions at a late stage of macroautophagy (hereafter autophagy). The Spin/Spns1 defect induces aberrant autolysosome formation that leads to developmental senescence in the embryonic stage and premature aging symptoms in adulthood. However, the molecular mechanism by which loss of Spin/Spns1 leads to the specific pathogenesis remains to be elucidated...
November 22, 2016: Autophagy
https://www.readbyqxmd.com/read/27866707/a-functional-variant-associated-with-atrial-fibrillation-regulates-pitx2c-expression-through-tfap2a
#6
Jiangchuan Ye, Nathan R Tucker, Lu-Chen Weng, Sebastian Clauss, Steven A Lubitz, Patrick T Ellinor
The most significantly associated genetic locus for atrial fibrillation (AF) is in chromosomal region 4q25, where four independent association signals have been identified. Although model-system studies suggest that altered PITX2c expression might underlie the association, the link between specific variants and the direction of effect on gene expression remains unknown for all four signals. In the present study, we analyzed the AF-associated region most proximal to PITX2 at 4q25. First, we identified candidate regulatory variants that might confer AF risk through a combination of mammalian conservation, DNase hypersensitivity, and histone modification from ENCODE and the Roadmap Epigenomics Project, as well as through in vivo analysis of enhancer activity in embryonic zebrafish...
December 1, 2016: American Journal of Human Genetics
https://www.readbyqxmd.com/read/27846271/igg-containing-isoforms-of-neuregulin-1-are-dispensable-for-cardiac-trabeculation-in-zebrafish
#7
Leigh Ann Samsa, Cade Ellis Ito, Daniel Ross Brown, Li Qian, Jiandong Liu
The Neuregulin-1 (Nrg1) signaling pathway has been widely implicated in many aspects of heart development including cardiac trabeculation. Cardiac trabeculation is an important morphogenetic process where clusters of ventricular cardiomyocytes extrude and expand into the lumen of the ventricular chambers. In mouse, Nrg1 isoforms containing an immunoglobulin-like (IgG) domain are essential for cardiac trabeculation through interaction with heterodimers of the epidermal growth factor-like (EGF-like) receptors ErbB2/ErbB4...
2016: PloS One
https://www.readbyqxmd.com/read/27845735/deletion-of-pr130-interrupts-cardiac-development-in-zebrafish
#8
Jie Yang, Zuhua Li, Xuedong Gan, Gang Zhai, Jiajia Gao, Chenling Xiong, Xueping Qiu, Xuebin Wang, Zhan Yin, Fang Zheng
Protein phosphatase 2 regulatory subunit B, alpha (PPP2R3A), a regulatory subunit of protein phosphatase 2A (PP2A), is a major serine/threonine phosphatase that regulates crucial function in development and growth. Previous research has implied that PPP2R3A was involved in heart failure, and PR130, the largest transcription of PPP2R3A, functioning in the calcium release of sarcoplasmic reticulum (SR), plays an important role in the excitation-contraction (EC) coupling. To obtain a better understanding of PR130 functions in myocardium and cardiac development, two pr130-deletion zebrafish lines were generated using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system...
November 11, 2016: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/27836208/zebrafish-genome-engineering-using-the-crispr-cas9-system
#9
REVIEW
Mingyu Li, Liyuan Zhao, Patrick S Page-McCaw, Wenbiao Chen
Geneticists have long sought the ability to manipulate vertebrate genomes by directly altering the information encoded in specific genes. The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonuclease has the ability to bind single loci within vertebrate genomes and generate double-strand breaks (DSBs) at those sites. These DSBs induce an endogenous DSB repair response that results in small insertions or deletions at the targeted site. Alternatively, a template can be supplied, in which case homology-directed repair results in the generation of engineered alleles at the break site...
December 2016: Trends in Genetics: TIG
https://www.readbyqxmd.com/read/27833165/genetic-and-pharmacological-inhibition-of-cdk9-drives-neutrophil-apoptosis-to-resolve-inflammation-in-zebrafish-in-vivo
#10
Laura J Hoodless, Christopher D Lucas, Rodger Duffin, Martin A Denvir, Christopher Haslett, Carl S Tucker, Adriano G Rossi
Neutrophilic inflammation is tightly regulated and subsequently resolves to limit tissue damage and promote repair. When the timely resolution of inflammation is dysregulated, tissue damage and disease results. One key control mechanism is neutrophil apoptosis, followed by apoptotic cell clearance by phagocytes such as macrophages. Cyclin-dependent kinase (CDK) inhibitor drugs induce neutrophil apoptosis in vitro and promote resolution of inflammation in rodent models. Here we present the first in vivo evidence, using pharmacological and genetic approaches, that CDK9 is involved in the resolution of neutrophil-dependent inflammation...
November 11, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27832146/ribozyme-mediated-grna-generation-for-in-vitro-and-in-vivo-crispr-cas9-mutagenesis
#11
Raymond Teck Ho Lee, Ashley Shu Mei Ng, Philip W Ingham
CRISPR/Cas9 is now regularly used for targeted mutagenesis in a wide variety of systems. Here we report the use of ribozymes for the generation of gRNAs both in vitro and in zebrafish embryos. We show that incorporation of ribozymes increases the types of promoters and number of target sites available for mutagenesis without compromising mutagenesis efficiency. We have tested this by comparing the efficiency of mutagenesis of gRNA constructs with and without ribozymes and also generated a transgenic zebrafish expressing gRNA using a heat shock promoter (RNA polymerase II-dependent promoter) that was able to induce mutagenesis of its target...
2016: PloS One
https://www.readbyqxmd.com/read/27829120/crispr-guide-rna-validation-in-vitro
#12
Stephanie Grainger, Brianna Lonquich, Chet Huan Oon, Nicole Nguyen, Karl Willert, David Traver
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has been applied to edit genomes in a wide variety of model systems. Although this process can be quite efficient, editing at precise locations in the genome remains difficult without a suitable single guide RNA (sgRNA). We have developed a method for screening sgRNA function in vitro, using reagents that most zebrafish laboratories are already using. The results from our in vitro assay correlate with function in vivo in every sgRNA that we have examined so far...
November 9, 2016: Zebrafish
https://www.readbyqxmd.com/read/27819330/stathmin-like-4-is-critical-for-the-maintenance-of-neural-progenitor-cells-in-dorsal-midbrain-of-zebrafish-larvae
#13
Meng-Ju Lin, Shyh-Jye Lee
A delicate balance between proliferating and differentiating signals is necessary to ensure proper growth and neuronal specification. By studying the developing zebrafish brain, we observed a specific and dynamic expression of a microtubule destabilizer gene, stathmin-like 4 (stmn4), in the dorsal midbrain region. The expression of stmn4 was mutually exclusive to a pan-neuronal marker, elavl3 that indicates its role in regulating neurogenesis. We showed the knockdown or overexpression of stmn4 resulted in premature neuronal differentiation in dorsal midbrain...
November 7, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27809318/a-high-throughput-functional-genomics-workflow-based-on-crispr-cas9-mediated-targeted-mutagenesis-in-zebrafish
#14
Gaurav K Varshney, Blake Carrington, Wuhong Pei, Kevin Bishop, Zelin Chen, Chunxin Fan, Lisha Xu, Marypat Jones, Matthew C LaFave, Johan Ledin, Raman Sood, Shawn M Burgess
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...
December 2016: Nature Protocols
https://www.readbyqxmd.com/read/27807677/crispr-cas9-in-zebrafish-an-efficient-combination-for-human-genetic-diseases-modeling
#15
REVIEW
Jiaqi Liu, Yangzhong Zhou, Xiaolong Qi, Jia Chen, Weisheng Chen, Guixing Qiu, Zhihong Wu, Nan Wu
The next-generation sequencing identifies a growing number of candidate genes associated with human genetic diseases, which inevitably requires efficient methods to validate the causal links between genotype and phenotype. Recently, zebrafish, with sufficiently high-throughput capabilities, has become a favored option to study human pathogenesis. In addition, CRISPR/Cas9-based approaches have radically reduced the efforts to introduce targeted genome engineering in various organisms. Here, we systemically review the basic considerations in the design of gene editing in zebrafish with CRISPR/Cas9, and explore the potential of the combination of these two to support efficient functional analysis of human genetic variants...
November 2, 2016: Human Genetics
https://www.readbyqxmd.com/read/27802373/innate-color-preference-of-zebrafish-and-its-use-in-behavioral-analyses
#16
Jong-Su Park, Jae-Ho Ryu, Tae-Ik Choi, Young-Ki Bae, Suman Lee, Hae Jin Kang, Cheol-Hee Kim
Although innate color preference of motile organisms may provide clues to behavioral biases, it has remained a longstanding question. In this study, we investigated innate color preference of zebrafish larvae. A cross maze with different color sleeves around each arm was used for the color preference test (R; red, G; green, B; blue, Y; yellow). The findings showed that 5 dpf zebrafish larvae preferred blue over other colors (B > R > G > Y). To study innate color recognition further, tyrosinase mutants were generated using CRISPR/Cas9 system...
October 2016: Molecules and Cells
https://www.readbyqxmd.com/read/27795824/loss-of-mgat5a-mediated-n-glycosylation-stimulates-regeneration-in-zebrafish
#17
Wuhong Pei, Sunny C Huang, Lisha Xu, Kade Pettie, María Laura Ceci, Mario Sánchez, Miguel L Allende, Shawn M Burgess
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...
2016: Cell Regeneration
https://www.readbyqxmd.com/read/27792783/deep-brain-photoreceptor-val-opsin-gene-knockout-using-crispr-cas-affects-chorion-formation-and-embryonic-hatching-in-the-zebrafish
#18
Chong Yee Hang, Shogo Moriya, Satoshi Ogawa, Ishwar S Parhar
Non-rod non-cone photopigments in the eyes and the brain can directly mediate non-visual functions of light in non-mammals. This was supported by our recent findings on vertebrate ancient long (VAL)-opsin photopigments encoded by the val-opsinA (valopa) and val-opsinB (valopb) genes in zebrafish. However, the physiological functions of valop isoforms remain unknown. Here, we generated valop-mutant zebrafish using CRISPR/Cas genome editing, and examined the phenotypes of loss-of-function mutants. F0 mosaic mutations and germline transmission were confirmed via targeted insertions and/or deletions in the valopa or valopb gene in F1 mutants...
2016: PloS One
https://www.readbyqxmd.com/read/27789478/microrna-126a-directs-lymphangiogenesis-through-interacting-with-chemokine-and-flt4-signaling-in-zebrafish
#19
Jian Chen, Rong-Fang Zhu, Fang-Fang Li, Yu-Lai Liang, Chen Wang, Yong-Wen Qin, Shuang Huang, Xian-Xian Zhao, Qing Jing
OBJECTIVE: MicroRNA-126 (miR-126) is an endothelium-enriched miRNA and functions in vascular integrity and angiogenesis. The application of miRNA as potential biomarker and therapy target has been widely investigated in various pathological processes. However, its role in lymphatic diseases had not been widely explored. We aimed to reveal the role of miR-126 in lymphangiogenesis and the regulatory signaling pathways for potential targets of therapy. APPROACH AND RESULTS: Loss-of-function studies using morpholino oligonucleotides and CRISPR/Cas9 system showed that silencing of miR-126a severely affected the formation of parachordal lymphangioblasts and thoracic duct in zebrafish embryos, although their development in miR-126b knockdown embryos was normal...
October 27, 2016: Arteriosclerosis, Thrombosis, and Vascular Biology
https://www.readbyqxmd.com/read/27777301/forkhead-transcription-factor-3a-foxo3a-modulates-hypoxia-signaling-via-up-regulation-of-the-von-hippel-lindau-gene-vhl
#20
Xing Liu, Xiaolian Cai, Bo Hu, Zhichao Mei, Dawei Zhang, Gang Ouyang, Jing Wang, Wei Zhang, Wuhan Xiao
FOXO3a, a member of the forkhead homeobox type O (FOXO) family of transcriptional factors, regulates cell survival in response to DNA damage, caloric restriction, and oxidative stress. The von Hippel-Lindau (VHL) tumor suppressor gene encodes a component of the E3 ubiquitin ligase complex that mediates hypoxia-inducible factor α degradation under aerobic conditions, thus acting as one of the key regulators of hypoxia signaling. However, whether FOXO3a impacts cellular hypoxia stress remains unknown. Here we show that FOXO3a directly binds to the VHL promoter and up-regulates VHL expression...
December 2, 2016: Journal of Biological Chemistry
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