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

• 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
• 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

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 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 流体的运动。已经描述了通过纤毛调节多种信号传导途径， 包括Hedgehog、Wnt、Notched和PDGF。影响睫状体成分的突变与 许多人类疾病，统称为纤毛病。睫状体炎引起的疾病 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、多囊肾和癌症。纤毛病包括罕见的人类 疾病，例如但不限于Meckel综合征（MKS）、Joubert综合征（JBTS）、肾痨 （NPHP）和Bardet-Biedl综合征（BBS）。为了治疗这些疾病和紊乱， 了解纤毛功能障碍如何导致疾病的病理学。虽然许多建筑 纤毛方面的研究可以在细胞培养中进行，要了解病理学需要动物模型。 斑马鱼是研究人类疾病和纤毛病的有力模型;然而，它们一直被认为是一种新的动物。 历史上仅限于吗啉代敲除。我们最近在纤毛中产生了一些合子敲除 相关基因，并发现了一些疾病表型。然而，这些淘汰赛中有许多是 成体疾病表型的胚胎致死预防分析。对于这种类型的分析，需要 斑马鱼条件性基因敲除此外，条件性敲除还将提供一种工具， 分析一种表型的起源细胞或在多种表型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， 制作起来很麻烦我们假设使用CRISPR/Cas9介导的可逆FlEx基因的敲入， 破坏盒我们将产生ift 88、mks 5和bbs 5条件等位基因。我们将在 在允许的方向上，它们像野生型等位基因一样起作用，而在非允许的方向上，它们像空等位基因一样起作用。 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