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Generate Zebrafish Conditional Knockout Model for Ciliopathy Research

生成用于纤毛病研究的斑马鱼条件敲除模型

基本信息

批准号：
10302461
负责人：
John M Parant
金额：
$ 22.28万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-15 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10302461
关键词：
Address Adult Affect Alleles Animal Model Anosmia Bardet-Biedl Syndrome Biological Models CRISPR/Cas technology Cell Culture Techniques Cell surface Cells Cilia Communities Complex Cystic kidney DNA Data Defect Disease Disease Progression Drug Screening Embryo Environment Erinaceidae Event Exons Fertilization Foundations Functional disorder Generations Genes Genetic Genetic Recombination Genetic Screening Genome engineering Heart Abnormalities Human Hydrocephalus Institution Introns Joubert syndrome Knock-in Knock-out Knowledge Lead Left Liquid substance Mechanics Meckel-Gruber syndrome Mediating Medical Microinjections Microtubules Modeling Morphology Movement Mutation National Institute of Arthritis and Musculoskeletal and Skin Diseases National Institute of Diabetes and Digestive and Kidney Diseases National Institute of General Medical Sciences National Institute on Deafness and Other Communication Disorders Nephronophthisis Neural Tube Closure Obesity Organelles Pathology Pathway interactions Phenotype Platelet-Derived Growth Factor Play Polycystic Kidney Diseases Polydactyly RNA Reagent Regulation Renal carcinoma Reporter Genes Research Research Design Retinal Degeneration Role Signal Pathway Signal Transduction Site Spinal Curvatures Structure Study models Time Tissues United States National Institutes of Health Visualization Zebrafish aqueous base ciliopathy conditional knockout disease phenotype embryonic stem cell experimental study genome editing hair cell regeneration high reward high risk human disease knock-down knockout gene man mutant notch protein prevent protein transport scoliosis tool vesicle transport

项目摘要

Abstract Cilia are evolutionarily conserved microtubule-based organelles that project from the surface of the cells and plays an important role in protein trafficking, signaling cascade regulation, and mechanical movement of fluids. Multiple signaling pathways have been described to be regulated through the cilia, including Hedgehog, Wnt, Notched, and PDGF. Mutations that affect ciliary components are associated with a multitude of human diseases, together called ciliopathies. The medical conditions that result from ciliary dysfunction include, but are not limited to, retinal degeneration, anosmia, neural tube closure, polydactyly, cardiac malformations, obesity, polycystic kidney disease and cancer. Ciliopathies include rare human disorders such as, but not limited to, Meckel Syndrome (MKS), Joubert Syndrome (JBTS), Nephronophthisis (NPHP), and Bardet-Biedl Syndrome (BBS). With hopes of treating these diseases and disorders it is important understand how ciliary dysfunction contributes to the pathology of disease. While many of the structural aspects of cilia can be studies in cell culture, to understand the pathology animal models are required. Zebrafish is a powerful model for studying human disease and ciliopathies; however, they have been historically limited to morpholino knockdown. We have recently generated a number zygotic knockout in cilia associated genes, and discovered a number of disease phenotypes. However many of these knockouts are embryonic lethal preventing analyses of adult disease phenotypes. For this type of analysis, there is a need for zebrafish conditional knockouts. Further, a conditional knockout would also provide a tool for embryonic analysis as far as cell of origin of a phenotypes or isolation of one phenotype outside the context of multiple phenotypes. However, the generation of zebrafish conditional alleles are technically challenging and cumbersome to make. We hypothesis that using CRISPR/Cas9 mediated knock-in of an invertible FlEx gene disruption cassette we will generate ift88, mks5, and bbs5 conditional allele. We will validate that in the permissive orientation they act like a wild type allele, and in the non-permissive orientation, they act like a null allele. Further, we will validate the ability to induce temporal and spatial Cre induced recombination with these alleles. Successful completion of this proposal will provide a highly useful animal model to the research community.

摘要