Mouse models for the functional analysis of asthma-associated human polymorphisms

用于哮喘相关人类多态性功能分析的小鼠模型

• 批准号: 7873363
• 负责人: Donata Vercelli
• 金额: $ 4.01万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-18 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7873363
• 关键词: 3' Untranslated Regions; 5' Flanking Region; Allergens; Allergic; Animals; Arginine; Asthma; Atopic Dermatitis; Bacterial Artificial Chromosomes; Biological; Bone Marrow; Caucasians; Caucasoid Race; Cells; Code; Complex; DNA Resequencing; Data; Data Reporting; Development; Disease; Distal; Elements; Ensure; Event; Exhibits; Exons; Functional RNA; Gene Cluster; Gene Expression; Generations; Genes; Genetic; Genetic Determinism; Genetic Heterogeneity; Genetic Polymorphism; Genetic Transcription; Genetic Variation; Genomics; Glutamine; Goals; Hand; Haplotypes; Human; Hypersensitivity; IL4 gene; IL5 gene; In Vitro; Individual; Inflammation; Interleukin-13; Introns; Knock-out; Knockout Mice; Lead; Link; Lung; Lung Inflammation; Mediating; Messenger RNA; Mitogens; Modeling; Molecular; Mononuclear; Mus; Pathogenesis; Patients; Pattern; Phenotype; Physiological; Play; Population; Positioning Attribute; Post-Transcriptional Regulation; Predisposition; Proteins; Regulation; Regulatory Element; Relative (related person); Research; Risk; Role; Secondary to; Serum; Single Nucleotide Polymorphism; Testing; Th2 Cells; Tissues; Transgenes; Transgenic Mice; Transgenic Organisms; Variant; Work; airway hyperresponsiveness; base; cis acting element; cytokine; falls; gene function; genetic analysis; genetic manipulation; genetic variant; in vivo; in vivo Model; interest; mast cell; mouse model; novel; overexpression; prevent; promoter; protein expression; public health relevance; response; skills; tool; trait

DESCRIPTION (provided by applicant): Asthma and allergy are marked by a profound dysregulation of Th2 responses and increased expression of the Th2 cytokines IL5, IL13 and IL4. Genetic variants in the Th2 cytokine locus are strongly associated with allergic inflammation, but their impact on the phenotype is far from understood because the genetic heterogeneity and complexity existing within outbred populations prevent association studies from revealing which genetic variants are involved in phenotype determination. Our goal is to characterize the mechanism(s) underlying the impact of natural variants on the expression and function of genes critical for the development of, and the susceptibility to, human allergic inflammation. Our model gene is IL13, a key effector in asthma and allergy. We showed that: (1) IL13 variants are strong determinants of asthma/allergy; (2) the IL13 locus includes two blocks of highly linked single nucleotide polymorphisms (SNP); and (3) IL13+2044G>A in the 3' block, results in the expression of an IL13 R130Q variant more active than wild type (WT) IL13, whereas IL13-1112C>T in the 5' block increases IL13 transcription in Th2 cells and IL13 secretion ex vivo. Despite these advances, we are convinced ultimately regulatory SNPs need to be studied in vivo within a physiologic genomic context. Here we wish to explore the hypothesis that the impact of genetic variation on human IL13 regulation can be effectively modeled and dissected in BAC transgenic (TG) mice carrying WT or asthma/allergy-associated human IL13 haplotypes. More specifically, we propose: Specific Aim 1: To generate and validate [murine IL13-deficient] BAC TG mouse lines carrying the WT human IL13 locus or the IL13 haplotypes most commonly associated with asthma traits in Caucasians. This work will capitalize on our analysis of variation in IL13 and our skills in BAC recombineering, and will lead to the generation of TG lines exhibiting faithful tissue-specific and copy number-dependent expression of human IL13, but lacking murine IL13. Specific Aim 2: To identify functional IL13 polymorphisms, and the underlying mechanisms, by comparing and contrasting human IL13 regulation and IL13-dependent lung responses in [murine IL13-deficient] BAC TG lines carrying distinct IL13 haplotypes. This work will determine whether variation acts on IL13 transcriptional and/or post-transcriptional regulation or through the secretion of an overactive IL13 protein variant (IL13 R130Q). [The murine IL13-deficient background will be ideal for the characterization of human IL13-dependent phenotypes]. By providing a controlled genetic background, this in vivo model will define the SNPs (or blocks thereof) involved in IL13 dysregulation and their modifying effects on IL13 expression and/or function, paving the way for successful strategies to neutralize the effects of genetic dysregulation in IL13-mediated disease. PUBLIC HEALTH RELEVANCE. The overall goal of our work is to characterize the mechanisms underlying the impact of natural genetic variation on the expression and function of genes critical for the development of, and the susceptibility to, human allergic inflammation. Our model is IL13, a Th2 cytokine which is overexpressed in patients with asthma and/or allergy. We have studied variation in IL13 quite extensively, and we have identified several polymorphisms that dysregulate the expression and/or the function of this gene in vitro. Clearly, a genetically determined increase in IL13 expression and/or activity is likely to play a major role in the pathogenesis of asthma and allergy. Despite these advances, we are convinced novel, more powerful and physiologic approaches are required to elucidate the role played by genetic variation in IL13 dysregulation and IL13- mediated disease. Ultimately, polymorphisms need to be studied in vivo within the physiologic genomic context. In this proposal we wish to explore the hypothesis that (a) human IL13 polymorphisms associated with asthma-related phenotypes are sufficient to induce appreciable dysregulation of IL13 expression and/or function, and (b) the impact of natural genetic variation on human IL13 regulation can be effectively modeled and dissected in mouse models carrying defined wild type or asthma/allergy-associated human IL13 haplotypes [on a murine IL13- deficient background]. By ensuring haplotype-specific patterns of IL13 regulation are gauged against a controlled genetic background, this in vivo model will allow us to determine which polymorphisms are necessary and sufficient for IL13 dysregulation, leading to the molecular mechanisms responsible for altered IL13 expression and/or function.

描述（由申请人提供）：哮喘和过敏是TH2反应的深刻失调以及Th2细胞因子IL5，IL13和IL4的表达增加。 Th2细胞因子基因座中的遗传变异与过敏性炎症密切相关，但是它们对表型的影响远未理解，因为在胞本群体中存在的遗传异质性和复杂性阻止关联研究揭示哪些遗传变异与表型有关。我们的目标是表征自然变异对人类过敏性炎症至关重要的基因表达和功能的影响的基础机制。我们的模型基因是IL13，这是哮喘和过敏的关键效应子。我们表明：（1）IL13变体是哮喘/过敏的强决定因素； （2）IL13基因座包括两个高度连接的单核苷酸多态性（SNP）的块； （3）IL13+2044G>在3'块中，导致IL13 R130Q变体的表达比野生型（WT）IL13更活跃，而5'块中IL13-1112C> t的表达会增加Th2细胞和IL13 Cellion的IL13转录。尽管取得了这些进步，但我们确信最终需要在生理基因组环境中在体内研究调节性SNP。在这里，我们希望探讨以下假设：遗传变异对人IL13调节的影响可以有效地建模和解剖，该遗传转基因（TG）小鼠携带WT或哮喘/过敏相关的人IL13单倍型。更具体地说，我们提出：特定目的1：生成和验证携带WT人IL13基因座或IL13单倍型的BAC TG小鼠线最常见的是与高加索人中哮喘性状相关的IL13单倍型。这项工作将利用我们对IL13的变异分析以及我们在BAC重新组合方面的技能，并将导致TG系的产生，表现出忠实的组织特异性和副本依赖人IL13的表达，但缺乏鼠IL13。具体目的2：通过比较和对比[鼠IL13缺陷型] BAC TG系中的人类IL13调节和IL13依赖性肺反应，以鉴定功能性IL13多态性和潜在机制。这项工作将确定变异是对IL13转录和/或转录后调控的作用，还是通过分泌过度活跃的IL13蛋白质变异（IL13 R130Q）。 [鼠IL13缺陷背景对于人类IL13依赖性表型的表征是理想的。通过提供受控的遗传背景，该体内模型将定义涉及IL13失调及其对IL13表达和/或功能的修改影响的SNP（或其块），从而为成功的策略铺平了途径，以中和中和IL13介导疾病中遗传失调的影响。公共卫生相关性。我们工作的总体目标是表征自然遗传变异对基因表达和功能对人类过敏性炎症至关重要的基因表达和功能的影响。我们的模型是IL13，这是一种Th2细胞因子，在哮喘和/或过敏的患者中过表达。我们已经非常广泛地研究了IL13的变化，并且已经确定了几种多态性，这些多态性在体外的表达和/或该基因的功能失调。显然，遗传确定的IL13表达和/或活性的增加可能在哮喘和过敏的发病机理中起主要作用。尽管取得了这些进步，但我们仍然需要新颖，更强大和生理方法来阐明IL13失调和IL13-介导的疾病在遗传变异中起作用的作用。最终，需要在生理基因组环境中的体内研究多态性。在这项提议中，我们希望探讨以下假设：（a）与哮喘相关表型相关的人类IL13多态性足以诱导IL13表达和/或功能的明显失调，以及（b）自然遗传变异对人类IL13调节的影响，在人类IL13调节中具有有效的模型或赋予的典型模型，是有效的，是在老鼠模型中划定的，是在老鼠类型中划定的，是在老鼠类型中划定的，是在老鼠类型中划定的典型模型。单倍型[在鼠IL13缺陷背景上]。通过确保对受控遗传背景的IL13调节的单倍型特异性模式进行测量，该体内模型将使我们能够确定哪些多态性是必需的，并且足以使IL13失调，从而导致分子机制负责改变IL13表达和/或功能的分子机制。