Automated Forward Genetic Analysis of Adaptive Immunity

项目总结 为了从机制上理解免疫，人们必须确定它的组成部分：那些与非 冗余功能在任何免疫过程中都是多余的，但我们选择给它下定义。人们可能会认为 大多数必需蛋白质都是已知的，但它们并不是。这一结论是基于不断增长的 我们实验室开发的一种哺乳动物遗传学新方法的经验。通过损坏或 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进行测序 突变，并在表型筛选之前对所有G3动物的潜在突变位点进行基因预分型，我们正在 能够实时确定哪些突变导致表型。实际上，我们现在有位置地克隆 瞬间：当一种表型被看到时，其原因就是已知的。我们也确切地知道我们有哪些基因 改变，做出了什么改变，以及对突变进行了多少次表型效应测试 纯合子状态。在提交这份提案之前的20个月里，我们对30,446人应用了这种方法 来自1189个家系的G3突变小鼠。每只突变小鼠都接受了110次免疫学检测。 测试17,640个基因中70,144个编码或剪接变化的免疫学效果。对于1,865个基因，在 在纯合状态下，至少检查了一个推定的零等位基因三次或更多次。我们估计我们 已经对基因组中18.4%的基因进行了严格的测试，以确定在免疫功能下降中发挥的必要作用 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对一种免疫学至关重要的蛋白质。 进程或其他进程，其中一些已知，但大多数以前未被识别1。这项初步调查 清楚地表明，关于人们可能认为研究得很好的现象，仍有许多有待了解的地方。何谓 T依赖抗体反应的要求是什么？我们可能认为我们知道，但实际上，许多 基本蛋白质仍未被发现。由于免疫系统的100多个新组件已经被 通过突变鉴定，我们有责任了解至少一些突变是如何 产生它们的表型效应。我们选择了解决基因的一个子集，在这些子集内，突变导致 与适应性免疫有关的显著表型。首先，我们将通过以下方式探讨过敏反应的基础 研究一组四个基因的突变导致对注射的木瓜酶和/或 卵清蛋白/明胶免疫。第二，我们将检查三种未知基因的突变。 与免疫有关，极大地影响淋巴细胞的发育和/或功能。第三，我们将研究 针对导致选择性高IGD综合征的突变而进行的IGD的调节和功能。我们会 还在继续诱变和筛选小鼠的其他免疫缺陷，并使我们的数据可供 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.