Automated Forward Genetic Analysis of Adaptive Immunity

适应性免疫的自动正向遗传分析

• 批准号: 10328571
• 负责人: BRUCE A BEUTLER
• 金额: $ 196.46万
• 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 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10328571
• 关键词: Affect; Alleles; Amino Acids; Antibody Response; B-Lymphocytes; Binding; Biological Assay; CRISPR/Cas technology; Candidate Disease Gene; Categories; Cell physiology; Cells; Code; Computer software; Data; Databases; Defect; Development; Elements; Ethylnitrosourea; Etiology; Fingerprint; Flow Cytometry; Funding; Genes; Genetic Screening; Genetic Transcription; Genetic study; Genome; Germ-Line Mutation; Immune; Immune System Diseases; Immune system; Immunity; Immunization; Immunoglobulin M; Immunologics; Immunology; Individual; Induced Mutation; Knock-out; Knowledge; Laboratories; Link; Lymphocyte; Lymphoid; Measurement; Measures; Meiosis; Messenger RNA; Metabolism; Methods; Molecular; Mus; Mutagenesis; Mutant Strains Mice; Mutate; Mutation; Names; Nonsense-Mediated Decay; Paper; Pathway interactions; Patients; Phenotype; Proteins; Publications; Publishing; RNA Processing; RNA Splicing; Recurrence; Regulation; Research Personnel; Role; Signal Transduction; Signaling Protein; Source; Supporting Cell; Surveys; Testing; Time; Transducers; Transfer RNA; Translations; Update; Validation; Vesicle; Work; adaptive immune response; adaptive immunity; causal variant; computerized tools; cytokine; gene discovery; gene product; genetic analysis; genetic pedigree; immune function; improved; insight; interest; loss of function mutation; mutation screening; novel; phenotypic data; protein complex; protein folding; receptor; response; screening; tool; trafficking; vesicle transport

PROJECT SUMMARY During the past four years, we have made outstanding progress in mutagenizing the mouse germline genome while keeping immunity under close surveillance. Now four years into the five-year project, we have thoroughly examined viable hypomorphic mutations in more than half of all protein-encoding genes. We have declared with high confidence that 1,298 mutations in 638 genes are causative of phenotypes in FACS and/or antibody response screens. Many of these genes were novel in that their necessity for immune function had been unknown, and were re-targeted by CRISPR/Cas9 to verify causation. 154 of the 638 candidate genes (24%; almost all of them novel) were either knocked out or modified with the ENU allele using CRISPR/Cas9 editing, expanded into pedigrees, and retested for causation of phenotypes detected in screening and automated mapping. 148 of the 154 genes (96%) were verified as the source of phenotype(s) declared. An additional 156 CRISPR/Cas9 projects are active at this time, some close to completion. Some of our discoveries have been published; many more are works in progress. However, all results of FACS assays on all mutations, whether declared causative of phenotype or not, have been de-restricted for public viewing on Mutagenetix, together with tools that enable search, examination of the original phenotypes and meiotic mapping data, filtering by P-value, and direction and magnitude of individual phenotypic effects. This will enable other laboratories to pursue mechanisms of immunological phenotypes alongside us. Knowledge of genes with non-redundant function in the development and activation of adaptive immune responses is fundamental to immunology and we plan to pursue screening further. We also plan deeper studies of the mechanism(s) behind phenotypes of particular interest. Of the phenotypes named for study in our earlier proposal, we have come to understand those caused by mutations in Trp53bp1, Ampd3, Rnps1, Prkd2, and Snrnp40, and have published papers describing mechanism. Additional phenotypes (not named in the original proposal) caused by mutations in Rabl3, Gpr89, Pdia6, Ncstn, Lmbr1l, Stk4, Pacs1, Wdr37, and Mfsd1 have also been elucidated and published or submitted for publication. We now propose to examine newly verified phenotypes, all the while creating novel phenotypes for study by ourselves and others. Our work is now guided by a tool (Similarity Heatmap) that measures relatedness of phenotypes. In flow cytometry screening, a minimum of 34 measurements are made from each mouse. The results constitute a phenotypic “fingerprint” amenable to tests of statistical similarity. Mutations in some genes yield results very similar to mutations in other genes, and we can sometimes infer that multiple genes operate within a single complex of proteins or enzymatic pathway. We have also written software (Candidate Explorer) to evaluate phenotypes in advance of declaring causation, telling us the likelihood of validation should we attempt to re-target any gene in question. We restrict our efforts to the most likely novel candidates, confident that all true causative relationships will ultimately manifest as saturation advances.

项目摘要 在过去的四年中，我们在小鼠生殖细胞基因组诱变方面取得了显著的进展 同时严密监视豁免权现在，五年计划已经进行了四年，我们已经彻底地 研究了超过一半的蛋白质编码基因中可行的亚型突变。我们已经宣布， 638个基因中1，298个突变是FACS和/或抗体中表型原因的高置信度 响应屏幕。这些基因中有许多是新的，因为它们对免疫功能的必要性已经被发现。 未知，并被CRISPR/Cas9重新靶向以验证因果关系。638个候选基因中有154个（24%; 几乎所有的都是新的）被敲除或使用CRISPR/Cas9编辑用ENU等位基因修饰， 扩展到家系，并重新检测筛选和自动化检测中检测到的表型的因果关系。 映射. 154个基因中的148个（96%）被证实为所声明的表型来源。新增156个 CRISPR/Cas9项目目前正在进行中，其中一些接近完成。我们的一些发现 出版;更多的是正在进行的工作。然而，所有突变的FACS测定结果，无论 宣布是否引起表型，已被解除限制，供公众在Mutagenetix上查看，以及 能够搜索、检查原始表型和减数分裂作图数据、通过P值过滤的工具， 以及个体表型效应的方向和大小。这将使其他实验室能够继续 免疫表型的机制。对非冗余功能基因的认识 适应性免疫反应的发展和激活是免疫学的基础，我们计划 进一步筛选。我们还计划深入研究特定表型背后的机制。 兴趣在我们先前的提议中命名为研究的表型中，我们已经了解了那些引起 通过Trp 53 bp 1、Ampd 3、Rnps 1、Prkd 2和Snrnp 40中的突变，并且已经发表了描述 机制由Rabl 3、Gpr 89突变引起的其他表型（原始提案中未命名）， Pdia 6、Nceptin、Lmbr 11、Stk 4、Pacs 1、Wdr 37和Mfsd 1也已阐明并发表或提交 出版。我们现在建议检查新验证的表型，同时创造新的表型 供自己和他人学习。我们的工作现在由一个工具（相似性热图）来指导， 表型的相关性。在流式细胞术筛选中，从每个细胞中进行至少34次测量。 老鼠.这些结果构成了一个表型“指纹”，可以进行统计相似性检验。突变 有些基因产生的结果与其他基因的突变非常相似，我们有时可以推断， 基因在蛋白质或酶途径的单一复合物内操作。我们还编写了软件 （候选探索者）在宣布因果关系之前评估表型，告诉我们 如果我们试图重新定位任何有问题的基因，我们只关注最有可能的小说 候选人，相信所有真正的因果关系将最终表现为饱和的进步。