Automated Forward Genetic Analysis of Adaptive Immunity

适应性免疫的自动正向遗传分析

• 批准号: 10623164
• 负责人: BRUCE A BEUTLER
• 金额: $ 196.23万
• 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/10623164
• 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; Maps; Measurement; Measures; Meiosis; Messenger RNA; Metabolism; Molecular; Mus; Mutagenesis; Mutant Strains Mice; Mutate; Mutation; Names; Nonsense-Mediated Decay; Paper; Pathway interactions; Patients; Phenotype; Probability; Productivity; 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; Writing; 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.

项目总结 在过去的四年里，我们在诱变小鼠生殖系基因组方面取得了显著的进展 同时在严密监视下保持豁免权。现在，五年计划已经进行了四年，我们已经彻底地 检查了超过一半的编码蛋白质的基因中有活性的亚型突变。我们已经声明了 638个基因的1,298个突变是FACS和/或抗体表型的原因，这一点具有高度的置信度 回复屏幕。这些基因中的许多都是新的，因为它们对免疫功能的必要性 不明，并被CRISPR/Cas9重新确定目标，以核实因果关系。在638个候选基因中有154个(24%； 几乎所有这些都是新的)使用CRISPR/Cas9编辑被敲除或用ENU等位基因修改， 扩展到家系，并重新测试筛查和自动化中检测到的表型的原因 映射。在154个基因中，有148个基因(96%)被证实为表型来源(S)。另外156人 CRISPR/CAS9项目目前正在进行中，有些项目接近完成。我们的一些发现是 已经出版；更多的作品正在进行中。然而，FACS的所有结果都是关于所有突变的，无论是 已在Mutagenetix上取消对公众查看的表型或非表型致病因素的限制，以及 能够搜索、检查原始表型和减数分裂图谱数据、按P值过滤、 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研究 免疫表型的机制和我们一起。人类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 进一步进行筛查。我们还计划对特殊表型背后的机制(S)进行更深入的研究 利息。在我们之前的提案中命名的表型中，我们已经理解了导致 通过Trp53bp1、Ampd3、Rnps1、Prkd2和Snrnp40的突变，并发表了描述 机制。由Rabl3，Gpr89， Pdia6、Ncstn、Lmbr1l、Stk4、Pacs1、Wdr37和Mfsd1也已被阐明并发表或提交 以供出版。我们现在建议检查新验证的表型，同时创造新的表型 供我们自己和他人学习。我们的工作现在由一个工具(相似性热图)指导，该工具可以测量 表型的关联性。在流式细胞术筛查中，每种方法至少要进行34次测量。 老鼠。这些结果构成了符合统计相似性检验的表型“指纹”。基因突变 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 基因在单一的蛋白质复合体或酶途径中起作用。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 如果我们尝试重新定位任何有问题的基因，验证。我们把我们的努力局限于最有可能的小说 候选人，相信所有真正的因果关系最终都会随着饱和度的提高而显现。

项目成果

The class I myosin MYO1D binds to lipid and protects against colitis.

I 类肌球蛋白 MYO1D 与脂质结合并预防结肠炎。

• DOI: 10.1242/dmm.035923
• 发表时间: 2018
• 期刊: Disease models & mechanisms
• 影响因子: 4.3
• 作者: McAlpine,William;Wang,Kuan-Wen;Choi,JinHuk;SanMiguel,Miguel;McAlpine,SarahGrace;Russell,Jamie;Ludwig,Sara;Li,Xiaohong;Tang,Miao;Zhan,Xiaoming;Choi,Mihwa;Wang,Tao;Bu,ChunHui;Murray,AnneR;Moresco,EvaMarieY;Turer,EmreE

Essential role of MFSD1-GLMP-GIMAP5 in lymphocyte survival and liver homeostasis.

MFSD1-GLMP-GIMAP5 在淋巴细胞存活和肝脏稳态中的重要作用。

• DOI: 10.1073/pnas.2314429120
• 发表时间: 2023
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Zhong,Xue;Moresco,JamesJ;Diedrich,JoleneK;Pinto,AntonioM;SoRelle,JeffreyA;Wang,Jianhui;Keller,Katie;Ludwig,Sara;Moresco,EvaMarieY;Beutler,Bruce;Choi,JinHuk
• 通讯作者: Choi,JinHuk

OVOL2 sustains postnatal thymic epithelial cell identity.

• DOI: 10.1038/s41467-023-43456-z
• 发表时间: 2023-11-27
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Zhong, Xue;Peddada, Nagesh;Wang, Jianhui;Moresco, James J;Zhan, Xiaowei;Shelton, John M;SoRelle, Jeffrey A;Keller, Katie;Lazaro, Danielle Renee;Moresco, Eva Marie Y;Choi, Jin Huk;Beutler, Bruce
• 通讯作者: Beutler, Bruce