Generate Zebrafish Conditional Knockout Model for Ciliopathy Research

摘要 纤毛是进化上保守的以微管为基础的细胞器，从 在蛋白质运输、信号级联调节和机械加工中起重要作用 液体的运动。已经描述了通过纤毛调节多个信号通路， 包括Hedgehog、WNT、Notch和PDGF。影响睫状体成分的突变与一种 人类的多种疾病，统称为纤毛病。由睫状毛引起的医疗状况 功能障碍包括但不限于视网膜变性、嗅觉障碍、神经管闭合、多指、 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，如但不限于，梅克尔综合征(MKS)、Joubert综合征(JBTS)、肾炎 (NPHP)和Bardet-Biedl综合征(BBS)。有希望治疗这些疾病和障碍，重要的是 了解睫毛功能障碍如何导致疾病的病理。虽然许多结构 纤毛可以在细胞培养方面进行研究，了解动物模型的病理学是必要的。 斑马鱼是研究人类疾病和纤毛疾病的有力模型；然而，它们一直是 历史上仅限于吗啡击倒。我们最近在纤毛中产生了一些合子基因敲除 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 成人疾病表型的胚胎致死预防分析。对于这种类型的分析，需要 斑马鱼有条件淘汰赛。此外，有条件的基因敲除也将为胚胎 一种表型的起源细胞的分析或在多个表型范围外的一种表型的分离 表型。然而，斑马鱼条件等位基因的产生在技术上是具有挑战性的 制造起来很笨拙。我们假设使用CRISPR/Cas9介导的可逆Flex基因的敲入 我们将生成ift88、mks 5和bbs5条件等位基因。我们将在 允许取向它们的行为就像野生型等位基因，而在非允许取向中，它们的行为就像零 等位基因。进一步，我们将验证诱导Cre诱导的时间和空间重组的能力 等位基因。这一方案的成功完成将为该研究提供一个非常有用的动物模型 社区。

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.