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: 7873361
• 负责人: Donata Vercelli
• 金额: $ 11.51万
• 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/7873361
• 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）：哮喘和过敏的特点是 Th2 反应严重失调以及 Th2 细胞因子 IL5、IL13 和 IL4 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，但它们对表型的影响尚不清楚，因为远交群体中存在的遗传异质性和复杂性阻碍了关联研究揭示哪些遗传变异参与表型决定。我们的目标是表征自然变异对人类过敏性炎症的发展和易感性至关重要的基因表达和功能的影响机制。我们的模型基因是 IL13，它是哮喘和过敏的关键效应因子。我们发现：(1) IL13 变异是哮喘/过敏的重要决定因素； (2) IL13基因座包含两个高度连锁的单核苷酸多态性(SNP)区块； (3) 3' 块中的 IL13+2044G>A 导致 IL13 R130Q 变体的表达比野生型 (WT) IL13 更活跃，而 5' 块中的 IL13-1112C>T 增加 Th2 中的 IL13 转录细胞和 IL13 分泌离体。尽管取得了这些进展，我们相信最终的调节性 SNP 需要在生理基因组背景下进行体内研究。在这里，我们希望探索这样的假设：遗传变异对人类 IL13 调节的影响可以在携带 WT 或哮喘/过敏相关人类 IL13 单倍型的 BAC 转基因 (TG) 小鼠中有效地建模和剖析。更具体地说，我们建议： 具体目标 1：生成并验证携带 WT 人 IL13 基因座或最常与白种人哮喘特征相关的 IL13 单倍型的[鼠 IL13 缺陷] BAC TG 小鼠系。这项工作将利用我们对 IL13 变异的分析以及我们在 BAC 重组工程方面的技能，并将产生 TG 系，这些 TG 系表现出人类 IL13 的忠实组织特异性和拷贝数依赖性表达，但缺乏鼠 IL13。具体目标 2：通过比较和对比携带不同 IL13 单倍型的[小鼠 IL13 缺陷]BAC TG 系中的人类 IL13 调节和 IL13 依赖性肺反应，确定功能性 IL13 多态性及其潜在机制。这项工作将确定变异是否作用于 IL13 转录和/或转录后调节，或者通过分泌过度活跃的 IL13 蛋白变体 (IL13 R130Q)。 [小鼠 IL13 缺陷背景将是表征人类 IL13 依赖性表型的理想选择]。通过提供受控的遗传背景，该体内模型将定义参与 IL13 失调的 SNP（或其模块）及其对 IL13 表达和/或功能的修饰作用，为中和遗传失调影响的成功策略铺平道路。 IL13介导的疾病。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人类过敏性炎症的发展和易感性至关重要的基因表达和功能的影响机制。我们的模型是 IL13，一种 Th2 细胞因子，在哮喘和/或过敏患者中过度表达。我们对 IL13 的变异进行了相当广泛的研究，并且我们已经鉴定了几种在体外失调该基因的表达和/或功能的多态性。显然，基因决定的 IL13 表达和/或活性增加可能在哮喘和过敏的发病机制中发挥重要作用。尽管取得了这些进展，我们相信需要新的、更强大的生理学方法来阐明遗传变异在 IL13 失调和 IL13 介导的疾病中所起的作用。最终，多态性需要在生理基因组背景下进行体内研究。在本提案中，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 调节的影响可以在携带特定野生型或哮喘/过敏相关人类 IL13 单倍型的小鼠模型中进行有效建模和解剖[在小鼠 IL13 缺陷背景下]。通过确保在受控遗传背景下测量 IL13 调节的单倍型特异性模式，该体内模型将使我们能够确定哪些多态性对于 IL13 失调是必要且充分的，从而导致 IL13 表达和/或功能改变的分子机制。