Genetic Analysis of Primary Immunodeficiency using Transgenic Zebrafish

描述（由申请人提供）：斑马鱼是研究脊椎动物疾病，特别是造血和免疫系统疾病的有力模型。该建议的主要目的是在斑马鱼中产生新的原发性免疫缺陷（PID）动物模型。更广泛的实验目标是使用这些模型来更深入地了解PID的分子基础，并识别具有指导适应性免疫正常功能的新功能的新基因或已知基因。为了实现这些目标，我们的策略是基于一种新的正向遗传筛选，使用我们以前创建的转基因斑马鱼系。在该系中，斑马鱼T细胞特异性p56lck启动子驱动eGFP表达（lck：eGFP），允许在任何发育阶段体内检测T细胞的不存在。这提供了一个很大的优势，在以前的屏幕上，幼虫必须福尔马林固定的淋巴细胞特异性基因的原位杂交检测。例如，在lck：eGFP背景下，可以在体内鉴定突变鱼，允许在其整个寿命期间进行感染测定和检查，以获得表型逆转和感染或自身免疫并发症。我们的实验室已经启动了这项筛查，我们已经分析了前150个家庭。这导致了6个突变株系的鉴定，这些突变株系在发育的不同阶段具有T细胞或胸腺缺陷。我们建议更详细地描述这些突变体，以加深我们对T细胞和胸腺发育中正常和异常途径的理解。我们将进行细胞自主性研究，以确定缺陷是否存在于T细胞或胸腺上皮细胞，并将继续绘制最有趣的突变体，以确定候选基因。我们还建议继续筛选更多的突变家族。我们以前已经确定了突变锡兰（cey）的独特组合缺乏T细胞，但正常的胸腺大小。此外，cey在胚胎肝脏发育的同等阶段缺乏造血干细胞。我们已经用紧密连锁的多态性标记定位了cey位点，并建议对其进行详细研究。cey突变体的克隆有望揭示一个先前未知功能的基因。这项工作将提供一套新的免疫缺陷脊椎动物，这将有助于阐明免疫和分子特征的PID。在未来，可以启动一系列的调查，包括：评估对不同病原体挑战的反应，并研究遗传修饰剂对人类疾病鱼类模型中疾病结果的影响。PID模型的使用与斑马鱼作为特别容易获得的发育模型系统的效用相结合，将产生对人类PID潜在的分子机制的新见解，最终使患有这些危及生命的疾病的患者受益。公共卫生相关性：为了进一步了解原发性免疫缺陷，并开发更好的诊断工具，需要开发相关的小动物模型。斑马鱼是一种小型脊椎动物，它能像人类一样产生免疫细胞，因此可以通过基因突变来模仿人类的免疫缺陷。在这项提案中，我们打算生产突变斑马鱼线，作为免疫缺陷模型，并将研究这些模型，以提高诊断和我们对人类免疫缺陷疾病的理解。

DESCRIPTION (provided by applicant): The zebrafish represents a powerful model for the study of vertebrate diseases, particularly those of the hematopoietic and immune systems. The primary objective of this proposal is to generate new animal models of primary immunodeficiency (PID) in the zebrafish. The broader experimental goal is to use these models to gain a deeper understanding of the molecular basis of PID and to identify novel genes or known genes with novel functions that direct normal functioning of adaptive immunity. To achieve these goals our strategy is based on a novel forward genetic screen using a transgenic line of zebrafish we have previously created. In this line the zebrafish T cell specific p56lck promoter drives eGFP expression (lck:eGFP), allowing for in vivo detection of absence of T cells at any stage of development. This provides a great advantage over previous screens where larvae had to be formalin fixed for detection of lymphocyte-specific genes by in situ hybridization. For example, on the lck:eGFP background mutant fish can be identified in vivo, permitting infection assays and examination throughout their life-span for phenotypic reversion and infectious or autoimmune complications. This screen has already been initiated in our laboratory and we have analyzed the first 150 families. This has led to the identification of 6 mutant lines with T cell or thymus defects that occur at different stages of development. We propose to characterize these mutants in more detail to deepen our understanding of normal and aberrant pathways in T cell and thymus development. We will carry out cell- autonomy studies to determine if the defect lies in T cells or thymic epithelial cells, and will go on to map the most interesting mutants to identify candidate genes. We also propose to continue screening more mutant families. We have previously identified the mutant ceylon (cey) with the unique combination of absence of T cells but normal thymus size. In addition, cey lacks hematopoietic stem cells at the fetal liver equivalent stage of development. We have mapped the cey locus with closely linked polymorphic markers and propose to study it in detail. Cloning of the cey mutant promises to reveal a gene with previously unknown function. The work laid out in this proposal will provide a novel set of immunodeficient vertebrates that will be instrumental in elucidating immunological and molecular characteristics of PIDs. In the future a series of investigations can be initiated, including: evaluating response to different pathogenic challenges, and studying the influence of genetic modifiers on disease outcomes in fish models of human disease. The use of PID models coupled with the utility of zebrafish as a particularly accessible developmental model system will yield new insights into the molecular mechanisms underlying human PIDs, ultimately benefiting patients afflicted with these life- threatening diseases. PUBLIC HEALTH RELEVANCE: To further our understanding of primary immunodeficiencies, and to develop better diagnostic tools, relevant small animal models need to be developed. The zebrafish is a small vertebrate animal that produces immune cells much like humans and can therefore be used to imitate human immunodeficiencies by mutating its genes. In this proposal we intend to produce mutant zebrafish lines that serve as models for immunodeficiency, and will study these models to improve the diagnosis and our understanding of human immunodeficiency disorders.