Zebrafish Resource Core

斑马鱼资源核心

基本信息

批准号：
10213224
负责人：
LILIANNA SOLNICAKREZEL
金额：
$ 8.96万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-15 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10213224
关键词：
Adult Alleles Animal Model Animals Antibodies Bioinformatics CRISPR/Cas technology Caenorhabditis elegans Cells Code Collection Communities DNA Defect Development Diagnosis Dideoxy Chain Termination DNA Sequencing Disease Disease model Embryo Embryonic Development Evaluation Exhibits Experimental Models Fertility Fertilization Fishes Gene Expression Generations Genes Genomic DNA Genomics Guide RNA Human Image Injections International Invertebrates Knock-in Laboratories Larva Leadership Maintenance Methods Microscopy Modeling Morphology Mutation Oligonucleotides Optics Organ Organism Orthologous Gene Pathogenicity Patients Phenotype Protein Sequence Analysis Proteins RNA Reporting Resources Robotics Scheme Skeleton Structure Symptoms Testing Time Tissues Universities Variant Washington Zebrafish Zebrafish Proteins base deep sequencing design experimental study feeding gene product genetic analysis genetic variant genome editing homologous recombination human disease improved in vivo insertion/deletion mutation interest loss of function male mature animal medical schools microCT mutant mutation carrier overexpression rapid growth reverse genetics screening soft tissue spatiotemporal sperm cell transcription activator-like effector nucleases transmission process zebrafish development zebrafish genome zygote

Adult Alleles Animal Model Animals Antibodies Bioinformatics CRISPR/Cas technology Caenorhabditis elegans Cells Code Collection Communities DNA Defect Development Diagnosis Dideoxy Chain Termination DNA Sequencing Disease Disease model Embryo Embryonic Development Evaluation Exhibits Experimental Models Fertility Fertilization Fishes Gene Expression Generations Genes Genomic DNA Genomics Guide RNA Human Image Injections International Invertebrates Knock-in Laboratories Larva Leadership Maintenance Methods Microscopy Modeling Morphology Mutation Oligonucleotides Optics Organ Organism Orthologous Gene Pathogenicity Patients Phenotype Protein Sequence Analysis Proteins RNA Reporting Resources Robotics Scheme Skeleton Structure Symptoms Testing Time Tissues Universities Variant Washington Zebrafish Zebrafish Proteins base deep sequencing design experimental study feeding gene product genetic analysis genetic variant genome editing homologous recombination human disease improved in vivo insertion/deletion mutation interest loss of function male mature animal medical schools microCT mutant mutation carrier overexpression rapid growth reverse genetics screening soft tissue spatiotemporal sperm cell transcription activator-like effector nucleases transmission process zebrafish development zebrafish genome zygote

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项目摘要

The Zebrafish Resource Core (Zf-Core) will be an integral component of the Washington University in St. Louis School of Medicine Model Organism Screening Center (wuMOSC), a multi-model organism platform for functional analysis of Undiagnosed Diseases Network (UDN) variants. It will serve as a resource for gene variant modeling, particularly in cases where analysis is not feasible in invertebrate model organisms, such as D. melanogaster and/or C. elegans. We anticipate that our bioinformatic analyses will designate ~65 variants per year to be analyzed in the zebrafish model. We will leverage the relatively short generation time, high fecundity, and external development of transparent zebrafish embryos to develop and implement a rapid, efficient, and disease variant-tailored pipeline for evaluating the variant pathogenicity using CRISPR/Cas9 approaches and/or RNA overexpression. The Zf-Core has improved on previous methods of fish husbandry by employing large-scale robotic feeding to allow for rapid growth of animals. This effectively halves the typical zebrafish generation time and thus will accelerate screening UDN variants proposed here. The PI laboratory also recently improved homologous recombination-based genome editing methods in zebrafish using TALEN or CRISPR/Cas9 strategies. For UDN gene-variants where the null allele manifests a phenotype prior to day 3 post fertilization, we will assess the degree to which the mutant phenotype can be rescued by injection into one-celled zygotes of synthetic RNA encoding the human protein variant, or zebrafish protein with this variant introduced. These experiments provide a rapid assessment as to whether the variant has normal, reduced, elevated or otherwise abnormal activity. If lines harboring nonsense or other deleterious mutations in the disease gene of interest already exist they will be imported to our Zf-Core. Alternatively, we will generate small insertions/deletions (indel) mutations using CRISPR/Cas9 strategy by designing a guide RNA that would also allow us to introduce the disease variant into the endogenous locus. In a second strategy, for genes with phenotypes detectable after day 3 post fertilization, the variant will be knocked-into the zebrafish ortholog by CRISPR/Cas9 editing. The phenotype of the resulting loss of function and knock-in mutants will be analyzed to verify any reported defects or to uncover them. Transparent embryos and larvae will be evaluated at the level of overall morphology, formation of specific organs and tissues using in vivo microscopy, and adults by microCT imaging to assess skeleton and soft tissues. Observation of a phenotype with a variant knock-in suggests that the variant is deleterious to the model organism and provides experimental evidence that it is a promising/likely disease-causing candidate. Lack of observable phenotype would indicate the experimental test provides no evidence for pathogenicity. The Zf- Core will be a strong component of the wuMOSC to inform the diagnosis of rare and poorly characterized diseases.

斑马鱼资源核心（ZF核）将是圣路易斯华盛顿大学不可或缺的组成部分医学学院模型有机体筛查中心（Wumosc），一个多模型生物平台未诊断疾病网络（UDN）变体的功能分析。它将作为基因的资源变体建模，特别是在无脊椎动物模型生物中分析不可行的情况下，例如 D. Melanogaster和/或秀丽隐杆线虫。我们预计我们的生物信息学分析将指定约65种变体每年要在斑马鱼模型中进行分析。我们将利用相对短期的时间，高繁殖力和外部发展透明的斑马鱼胚胎开发和实施快速，高效和疾病的变体使用CRISPR/CAS9方法和/或RNA过表达评估变异致病性的管道。 ZF核通过使用大规模的机器人喂养来改善了以前的鱼类饲养方法允许动物快速生长。这有效地将典型的斑马鱼生成时间切成一半，因此将加速此处提出的筛选UDN变体。 PI实验室最近还改善了同源使用TALEN或CRISPR/CAS9策略的斑马鱼基于重组的基因组编辑方法。对于UDN基因变异的，无效等位基因在受精后第3天表现出表型，我们将评估突变表型可以通过注射到一个单细胞合子的程度编码人类蛋白质变体或斑马鱼蛋白的合成RNA与该变体一起引入。这些实验提供了有关该变体是否正常，降低，升高或其他方式的快速评估异常活性。如果有疾病基因中有胡说八道或其他缺失突变的线它们已经存在，将进口到我们的ZF核心。另外，我们将生成小插入/删除（Indel）通过设计指南RNA使用CRISPR/CAS9策略的突变，该指南RNA也可以使我们引入疾病变体进入内源性基因座。在第二种策略中，对于受精后第3天可检测到表型的基因，这种变体将是通过CRISPR/CAS9编辑敲击斑马鱼直系同源物。由此产生的功能丧失的表型将分析敲入突变体，以验证任何报告的缺陷或发现它们。透明胚胎将在整体形态，特定器官和组织的形成水平上评估幼虫体内显微镜和通过MicroCT成像进行评估骨骼和软组织的成年人。观察具有变体敲入的表型表明该变体对模型有机体有害并提供实验性证据表明，这是一个有望/可能引起疾病的候选者。缺乏可观察的表型将表明实验测试没有提供致病性的证据。 ZF-核心将是强大的 Wumosc的组成部分，以告知罕见和特征性疾病较差的诊断。