Automated Forward Genetic Analysis of Adaptive Immunity

适应性免疫的自动正向遗传分析

• 批准号: 10209864
• 负责人: BRUCE A BEUTLER
• 金额: $ 196.36万
• 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/10209864
• 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，我们在诱变小鼠种系基因组方面取得了杰出进步 同时在密切监视下保持免疫力。现在进入五年项目四年了，我们已经彻底了 在所有蛋白质编码基因的一半以上中，检查了可行的低形态突变。我们已经宣布 高度信心对638个基因的1,298个突变是FACS和/或抗体中表型的属 响应屏幕。这些基因中有许多是新颖的，因为它们的免疫功能的必要性是 未知，并被CRISPR/CAS9重新定位以验证原因。 638个候选基因中的154个（24％； 几乎所有的小说）要么使用CRISPR/CAS9编辑用ENU等位基因敲定或修改 扩展为血统书，并因在筛查和自动化中检测到的表型而重新测试 映射。 154个基因中的148个（96％）被验证为表型的来源。另外156 CRISPR/CAS9项目目前很活跃，有些接近完成。我们的一些发现 出版；还有更多的工作正在进行中。但是，所有突变的FACS测定结果，是否 是否为表型宣布是为了公开观看诱变的限制 可以搜索，检查原始表型和减数分裂映射数据的工具，通过p值进行过滤， 以及单个表型效应的方向和大小。这将使其他实验室能够购买 与我们旁边的免疫表型的机制。了解具有非冗余功能的基因 适应性免疫回报的发展和激活是免疫学的基础，我们计划 进一步进行筛查。我们还计划对特定表型背后机制的更深入研究 兴趣。在我们较早提出的研究中命名的表型中，我们已经了解了那些引起的表型 通过TRP53BP1，AMPD3，RNPS1，PRKD2和SNRNP40中的突变，并发表了描述的论文 机制。由Rabl3中的突变引起的其他表型（未在原始提案中命名），GPR89， PDIA6，NCSTN，LMBR1L，STK4，PACS1，WDR37和MFSD1也已被阐明，出版或提交 出版。我们现在建议检查新验证的表型，同时创建新型表型 供我们自己和他人学习。现在，我们的工作以测量的工具（相似性热图）为指导 表型的相关性。在流式细胞仪筛查中，每个测量都至少进行34个测量 老鼠。该结果构成了适合统计相似性测试的表型“指纹”。突变 一些基因产生的结果与其他基因的突变非常相似，我们有时可以推断出多个 基因在蛋白质或酶促途径的单个复合物中起作用。我们还写了软件 （候选探险家）在宣布原因之前评估表型，并告诉我们 验证应试图重新靶向相关的任何基因。我们将努力限制为最有可能的小说 候选人，充满信心，所有真正的灾难性关系最终都将表现为满意度的进步。