Automated Forward Genetic Analysis of Adaptive Immunity

项目摘要 为了从机制上理解免疫，我们必须确定其组成部分：那些非免疫性的蛋白质。 在任何免疫过程的行使中，冗余功能，无论我们选择如何定义它。 大多数必需蛋白质是已知的，但它们不是。这一结论是基于增长 我们实验室开发的哺乳动物遗传学新方法的经验1。通过破坏或 用ENU随机破坏基因，对这些基因的每个G1男性携带者的整个外显子组进行测序， 突变，并在表型筛选前在潜在突变位点对所有G3动物进行预基因分型， 能够在真实的时间内确定哪些突变导致表型。实际上，我们现在定位克隆 瞬间：当一个表型被看到，它的原因是已知的。我们也知道我们有哪些基因 改变了，做了什么改变，以及在突变中测试了多少次表型效应。 纯合状态在提交本建议书之前的20个月里，我们对30，446人采用了这种方法。 来自1，189个家系的G3突变小鼠。对每只突变小鼠进行110次免疫测定 测试17,640个基因中70,144个编码或剪接变化的免疫学效应。对于1，865个基因， 在纯合状态下检查至少一个假定的无效等位基因三次或更多次。我们估计 严格测试了基因组中18.4%的基因，以确定这些基因在免疫功能下降中的必要作用。 在我们的兴趣范围内。在这样做的过程中，我们已经确定了许多蛋白质， 过程或另一个，其中一些已知的，但他们中的大多数以前不承认1。这次初步调查 它清楚地表明，对于人们可能认为研究得很好的现象，还有很多东西有待了解。是什么 T依赖性抗体反应的要求我们可能认为我们知道，但事实上， 重要的蛋白质仍未被发现。由于免疫系统的100多个新组成部分已经被 通过突变鉴定，我们有责任了解至少一些突变是如何发生的 产生它们的表型效应。我们选择了解决一个基因子集，其中突变导致 与适应性免疫有关的显著表型。首先，我们将探索过敏反应的基础， 研究一组四个基因，其中突变导致对注射木瓜蛋白酶的IgE反应放大，和/或 卵清蛋白/明矾免疫。其次，我们将研究三个基因的突变，这些基因通常不被认为是 与免疫相关，显著影响淋巴细胞发育和/或功能。第三，我们将研究 根据导致选择性高IgD综合征的突变，IgD的调节和功能。我们将 我们还将继续诱变和筛选小鼠的其他免疫缺陷，并使我们的数据可用于 科学界通过为此目的开发的软件。

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.