Genetic Analysis of Primary Immunodeficiency using Transgenic Zebrafish

使用转基因斑马鱼进行原发性免疫缺陷的遗传分析

• 批准号: 7534273
• 负责人: NIKOLAUS S TREDE
• 金额: $ 22.58万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-08 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7534273
• 关键词: Animal Model; Attention; Autoimmune Process; Biological Assay; Biological Models; Candidate Disease Gene; Cell Transplantation; Cells; Characteristics; Chemical Modifier; Cloning; Collection; Computer Systems Development; Coupled; Data; Defect; Depth; Detection; Development; Diagnosis; Diagnostic; Disease; Disease Outcome; Disease model; Endoderm; Family; Fetal Liver; Fishes; Fluorescence Microscopy; Formalin; Future; Generations; Genes; Genetic; Genetic Screening; Goals; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Human; Image; Immune; Immune System Diseases; Immune system; Immunity; Immunologic Deficiency Syndromes; In Situ Hybridization; Individual; Infection; Investigation; Jaw; Knowledge; Label; Laboratories; Larva; Life; Link; Location; Longevity; Lymphocyte; Maps; Modeling; Molecular; Molecular Genetics; Molecular and Cellular Biology; Mutate; Mutation; Numbers; Organogenesis; Pancytopenia; Pathway interactions; Patients; Phenotype; Polymorphic Microsatellite Marker; Population; Public Health; Science; Screening procedure; Series; Sorting - Cell Movement; Sri Lanka; Staging; Stem cells; Study models; T-Lymphocyte; Testing; Thymic epithelial cell; Thymus Gland; Transgenic Organisms; Vertebrates; Work; Zebrafish; base; cell motility; design; fetal; genetic analysis; hematopoietic tissue; human disease; improved; in vivo; insight; interest; mutant; novel; positional cloning; promoter; research study; response; sample fixation; size; tool

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.

描述（由申请人提供）：斑马鱼代表了脊椎动物疾病，特别是造血和免疫系统疾病研究的强大模型。本提案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，并识别指导适应性免疫正常功能的新基因或具有新功能的已知基因。为了实现这些目标，我们的策略是基于一种新的前向基因筛选，使用我们之前创造的转基因斑马鱼。在该细胞系中，斑马鱼T细胞特异性p56lck启动子驱动eGFP表达（lck:eGFP），允许在体内检测任何发育阶段T细胞的缺失。这提供了一个很大的优势，比以前的筛选，幼虫必须是福尔马林固定检测淋巴细胞特异性基因的原位杂交。例如，缺乏eGFP背景的突变鱼可以在体内识别，允许在其整个生命周期内进行感染分析和检查，以确定表型逆转和感染或自身免疫性并发症。这个筛选已经在我们的实验室开始，我们已经分析了前150个家庭。这导致鉴定出6个突变系，在不同的发育阶段发生T细胞或胸腺缺陷。我们建议更详细地描述这些突变，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研究，以确定缺陷是否存在于T细胞或胸腺上皮细胞中，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。我们之前已经确定了突变型锡兰（cey），其独特的组合缺乏T细胞，但胸腺大小正常。此外，在胎儿肝脏发育的同等阶段，cey缺乏造血干细胞。我们已经用紧密相连的多态性标记绘制了关键位点，并提出了对其进行详细研究的建议。克隆cey突变体有望揭示一种以前未知功能的基因。这项工作将提供一组新的免疫缺陷脊椎动物，这将有助于阐明pid的免疫学和分子特征。未来可以开展一系列研究，包括：评估对不同致病挑战的反应，以及研究基因修饰剂对人类疾病模型鱼类疾病结果的影响。PID模型的使用与斑马鱼作为一个特别容易获得的发育模型系统的效用相结合，将对人类PID的分子机制产生新的见解，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，它产生的免疫细胞与人类非常相似，因此可以通过基因突变来模仿人类的免疫缺陷。在本提案中，我们打算生产突变斑马鱼系作为免疫缺陷模型，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。