Automated Forward Genetic Analysis of Adaptive Immunity

适应性免疫的自动正向遗传分析

• 批准号: 9158963
• 负责人: BRUCE A BEUTLER
• 金额: $ 161.32万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-20 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9158963
• 关键词: Address; Affect; Alleles; Animals; Antibody Formation; Antibody Response; Binding; Biological Assay; Biology; Cells; Code; Communities; Data; Defect; Development; Elements; Employee Strikes; Ethylnitrosourea; Exercise; Genes; Genome; Genotype; Hyperimmunoglobulinemia D; IgE; Immune; Immune Cell Activation; Immune system; Immunity; Immunization; Immunoglobulin D; Immunologic Tests; Learning; Light; Lymphocyte; Mammalian Genetics; Maps; Methods; Mus; Mutagenesis; Mutant Strains Mice; Mutate; Mutation; Papain; Phenotype; Process; Production; Proteins; RNA Splicing; Regulation; Role; Site; Surveys; Testing; Thinking; Time; Work; adaptive immunity; allergic response; base; cell type; exome; experience; falls; gene function; genetic analysis; genetic pedigree; immune function; interest; male; mutation carrier; novel; ovalbumin-alum; response; screening; software development

PROJECT SUMMARY To understand immunity mechanistically, one must identify its constituent parts: those proteins with non- redundant function in the exercise of any immune process, however we choose to define it. One might assume that most of the essential proteins are known, but they are not. This conclusion is based on growing experience with a new method in mammalian genetics, developed in our laboratory1. By damaging or destroying genes at random with ENU, sequencing the whole exome of every G1 male carrier of these mutations, and pre-genotyping all G3 animals at potential mutation sites prior to phenotypic screening, we are able to determine which mutations cause phenotype in real time. In effect, we now positionally clone instantaneously: when a phenotype is seen, its cause is known. We also know precisely which genes we have altered, what change was made, and how many times the mutation was tested for phenotypic effects in the homozygous state. Over the 20 months prior to submission of this proposal, we applied this method to 30,446 G3 mutant mice from 1,189 pedigrees. Each mutant mouse was subjected to 110 immunological assays testing the immunological effects of 70,144 coding or splicing changes in 17,640 genes. For 1,865 genes, at least one putative null allele was examined three or more times in the homozygous state. We estimate that we have stringently tested 18.4% of all genes in the genome for a necessary role in those immune functions falling within our sphere of interest. In so doing, we have identified many proteins essential for one immunological process or another, some of them known, but most of them previously unrecognized1. This preliminary survey makes it clear that much remains to be learned about phenomena one might regard as well studied. What are the requirements for a T-dependent antibody response? We may think we know, but in fact, many of the essential proteins are still undiscovered. As more than 100 new components of the immune system have been identified through mutagenesis, it is incumbent upon us to understand how at least some of the mutations produce their phenotypic effects. We have chosen to address a subset of genes, within which mutations cause striking phenotypes bearing on adaptive immunity. First, we will probe the basis of allergic responses by studying a set of four genes in which mutations cause exaggerated IgE responses to injected papain and/or ovalbumin/alum immunization. Second, we will examine mutations in three genes not generally known to be associated with immunity that dramatically affect lymphocyte development and/or function. Third, we will study the regulation and function of IgD in light of a mutation that causes a selective hyper-IgD syndrome. We will also continue to mutagenize and screen mice for still other immune defects, and make our data accessible to the scientific community via software developed for this purpose.

项目摘要 要从机械上理解免疫力，必须识别其成分部分：那些具有非 - 的蛋白质 在行使任何免疫过程中的冗余功能，但是我们选择定义它。一个人可能会假设 大多数必需蛋白是已知的，但事实并非如此。这个结论是基于增长 在我们的实验室中开发的哺乳动物遗传学新方法的经验1。通过损害或 用ENU随机破坏基因，对这些G1雄性载体的整个外显子组进行测序 突变，并在表型筛选之前先在潜在突变位点进行所有G3动物，我们是 能够实时确定哪些突变导致表型。实际上，我们现在定位克隆 瞬间：当看到表型时，其原因是已知的。我们还确切地知道我们拥有哪些基因 改变了，发生了什么变化，以及在突变中测试了多少次表型效应 纯合状态。在提交该提案之前的20个月中，我们将此方法应用于30,446 来自1,189个血统的G3突变小鼠。每只突变小鼠接受110个免疫学测定 测试17,640个基因中70,144个编码或剪接变化的免疫学影响。对于1,865个基因，在 在纯合状态下，至少对一个假定的无效等位基因进行了三次或更多次检查。我们估计我们 严格测试了基因组中所有基因的18.4％，在这些免疫功能中发挥了必要的作用 在我们感兴趣的领域。这样，我们已经确定了许多免疫学必不可少的蛋白质 过程或其他过程，其中一些已知，但其中大多数以前未被认可1。这项初步调查 清楚地表明，关于一个人可能认为的现象还有很多待了解。什么是 对T依赖性抗体反应的要求？我们可能认为我们知道，但实际上，许多 基本蛋白质仍然未被发现。由于免疫系统的100多个新组件已经 通过诱变确定，我们有责任了解至少某些突变是如何 产生其表型效应。我们选择解决基因的一部分，其中突变引起 具有自适应免疫的惊人表型。首先，我们将通过 研究一组四个基因，其中突变引起对注射木瓜蛋白酶和/或 卵蛋白/明矾免疫。其次，我们将检查三个通常不知道的三个基因中的突变 与极大地影响淋巴细胞发育和/或功能的免疫有关。第三，我们将学习 根据导致选择性超级IGD综合征的突变，IgD的调节和功能。我们将 还将继续诱变和筛选小鼠以获得其他免疫缺陷，并使我们的数据可访问 通过软件开发的科学界为此目的开发。