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：
10447814
负责人：
John M Parant
金额：
$ 18.56万
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 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10447814
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 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.

抽象的纤毛是进化上保守的基于微管的细胞器，从细胞表面突出细胞并在蛋白质运输、信号级联调节和机械作用中发挥重要作用流体的运动。多种信号传导途径已被描述为通过纤毛进行调节，包括 Hedgehog、Wnt、Notched 和 PDGF。影响睫状成分的突变与多种人类疾病，统称为纤毛病。由睫状体引起的健康状况功能障碍包括但不限于视网膜变性、嗅觉丧失、神经管闭合、多指畸形、心脏畸形、肥胖、多囊肾病和癌症。纤毛病包括罕见的人类疾病，例如但不限于梅克尔综合征 (MKS)、朱伯特综合征 (JBTS)、肾痨（NPHP）和巴代-比德尔综合症（BBS）。怀着治疗这些疾病和病症的希望，重要的是了解纤毛功能障碍如何影响疾病的病理。虽然许多结构纤毛方面可以通过细胞培养进行研究，以了解需要动物模型的病理学。斑马鱼是研究人类疾病和纤毛病的强大模型；然而，他们已经历史上仅限于吗啉敲低。我们最近在纤毛中产生了一些合子敲除相关基因，并发现了许多疾病表型。然而，许多这样的淘汰赛都是成人疾病表型的胚胎致死预防分析。对于此类分析，需要斑马鱼条件性敲除。此外，有条件的基因敲除也将为胚胎提供一种工具。就表型的细胞起源进行分析或在多种背景之外分离一种表型表型。然而，斑马鱼条件等位基因的产生在技术上具有挑战性并且制作起来很麻烦。我们假设使用 CRISPR/Cas9 介导可逆 FlEx 基因的敲入我们将生成 ift88、mks5 和 bbs5 条件等位基因。我们将在在许可方向，它们的行为就像野生型等位基因，而在非许可方向，它们的行为就像无效等位基因等位基因。此外，我们将验证诱导时间和空间 Cre 诱导重组的能力等位基因。该提案的成功完成将为研究提供非常有用的动物模型社区。