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Automated Forward Genetic Analysis of Adaptive Immunity

适应性免疫的自动正向遗传分析

基本信息

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
项目类别：
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

项目摘要

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