Core C - Mutagenesis, Screening and Cryopreservation

核心 C - 诱变、筛选和冷冻保存

基本信息

批准号：
10642552
负责人：
Lucienne CHATENOUD
金额：
$ 62.42万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-06-13 至 2028-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10642552
关键词：
5 year old Affect Age Alleles Antigens Attention Autoantibodies Autoimmune Diabetes Autoimmune Diseases Base Pairing Beta Cell Breeding CRISPR/Cas technology Cells Child Clinic Code Complex Congenic Mice Cryopreservation DNA Data Databases Date of birth Detection Developed Countries Development Diabetes Mellitus Disease Ear Elements Embryo Environmental Risk Factor Ethylnitrosourea Etiology Exhibits Female Genes Genetic Genetic Drift Genome Genomic DNA Genotype Harvest Health Hemorrhage Heritability Housing Human Human Resources Hygiene Hyperglycemia Inbred NOD Mice Inbreeding Incidence Induced Mutation Infiltration Insulin-Dependent Diabetes Mellitus Ions Islets of Langerhans Joints Knock-out Link Maps Mediating Meiosis Modification Monitor Mononuclear Mouse Strains Mus Mutagenesis Mutagens Mutation Nitrosourea Compounds Pathogenesis Periodicals Phenotype Pre-Clinical Model Predisposition Prevention Quantitative Trait Loci RNA Splicing Recovery Research Personnel Role Siblings Susceptibility Gene T-Lymphocyte Tail Technology Therapeutic Time Transgenic Organisms Variant animation causal variant congenic cytokine data dissemination experience forward genetics genetic pedigree genetic technology germ free condition high risk human disease insulin secretion male mouse development novel phenotypic data prevent programs pup receptor reverse genetics screening sperm cell sperm cryopreservation web site

项目摘要

PROJECT SUMMARY/ABSTRACT The personnel in Core C have years of experience in breeding, housing and conducting experimentation with NOD mice, which develop spontaneous T1D under specific pathogen-free conditions. A very specific requirement for NOD mice is impeccable hygiene of the colony since minimal variations may affect T1D incidence in both female and male mice. A strict program of sibling inbreeding, storage of embryos from highly inbred stock, and periodic recovery of embryos from this stock is also necessary to prevent genetic drift of the type that led to the establishment of the NOD/NckH and NOD/NckL sublines. Core C will generate 350 pedigrees of NOD/NckH G3 mice modified by N-ethyl-N nitrosourea (ENU)-induced mutations. Pedigrees generated during the program will exhibit an average of approximately 60 germline coding/splicing mutations and include >80 G3 female mice. Each ENU-treated G0 male will be bred to NOD/NckH females to obtain 10-20 G1 male founders. These will be retro-orbitally bled to recover high quality DNA for Illumina sequencing, and tail cuttings will be used to isolate DNA from all G2 and G3 females for genotyping by Ion Torrent sequencing; Core B will perform sequencing. All female G3 pups will be monitored every other day for the development of T1D over a period of 40 weeks. Experimental observations will consist of first detection of glucosuria confirmed by hyperglycemia. For all mice ear tag numbers, birth dates, and phenotypic data will be uploaded to the Mutagenetix database, so that Kaplan-Meier analysis can be performed weekly to take account of new developments in every pedigree. We will perform similar phenotypic analyses of pedigrees carrying CRISPR/Cas9-targeted mutations generated in Project 2, for the purposes of verification. Preliminary data confirm that modification of the NOD/NckH T1D phenotype can be achieved, and causative mutations instantly mapped, fully supporting the discovery program we propose. We will perform extended monitoring (through 40 weeks of age) to detect all phenotypes including those of lesser magnitude, but we will gain time by identifying the strongest causative mutations early on. When each G1 male has completed G2 breeding, epididymal sperm will be harvested for cryopreservation and public distribution via the MMRRC; the mutations and their phenotypic effects will be made public on the Mutagenetix website. At times, verification of causation depends on placing a knockout allele in trans with the original ENU- induced allele. Therefore, as indicated, we will transfer cryopreserved sperm from Core C to Project 2, where germline re-targeting will take place in the NOD/NckH strain. The close interaction between Core C and Core B will include shipment of DNA at monthly intervals from Core C to Core B and uploading of phenotypic data on a weekly basis. This will guarantee the identification of many novel modifier mutations to fuel the activities of Project 1 and Project 2.

项目概要/摘要 Core C的人员拥有多年的饲养、饲养和实验经验 NOD 小鼠，在特定的无病原体条件下发生自发性 T1D。一个非常具体的 NOD 小鼠的要求是群体的卫生状况无可挑剔，因为最小的变化可能会影响 T1D 的发病率在雌性和雄性小鼠中。严格的兄弟近交计划，高度近交品种的胚胎储存，并且定期从该库存中回收胚胎也是必要的，以防止导致遗传漂变的类型 NOD/NckH 和 NOD/NckL 亚系的建立。 Core C将产生350个NOD/NckH血统 G3 小鼠经 N-乙基-N 亚硝基脲 (ENU) 诱导的突变修饰。程序期间生成的谱系将表现出平均约 60 个种系编码/剪接突变，并包括 >80 个 G3 雌性小鼠。每个 ENU 处理的 G0 雄性将与 NOD/NckH 雌性交配，以获得 10-20 个 G1 雄性创始人。这些将是轨道后放血以回收用于 Illumina 测序的高质量 DNA，尾部切割将用于分离来自所有 G2 和 G3 雌性的 DNA，通过 Ion Torrent 测序进行基因分型；核心 B 将执行排序。所有 G3 雌性幼崽将每隔一天接受一次监测，以了解 40 天内 T1D 的发展情况几周。实验观察将包括首次检测到由高血糖证实的糖尿。为了所有小鼠的耳标号、出生日期和表型数据都将上传到 Mutagenetix 数据库，以便卡普兰-迈耶分析可以每周进行一次，以考虑每个谱系的新发展。我们将对携带 CRISPR/Cas9 靶向突变的谱系进行类似的表型分析项目2，用于验证目的。初步数据证实 NOD/NckH T1D 的修改可以实现表型，并立即绘制致病突变，全力支持发现计划我们建议。我们将进行长期监测（直至 40 周龄）以检测所有表型，包括那些幅度较小的突变，但我们可以通过尽早识别最强的致病突变来赢得时间。什么时候每个G1雄性完成G2繁殖后，将采集附睾精子进行冷冻保存并公开通过 MMRRC 分发；突变及其表型效应将在 Mutagenetix 上公开网站。有时，因果关系的验证取决于将敲除等位基因与原始 ENU 反式放置诱导等位基因。因此，如前所述，我们将把冷冻精子从 Core C 转移到项目 2，其中种系重新靶向将在 NOD/NckH 菌株中发生。 Core C与Core B的紧密互动将包括每月将 DNA 从 Core C 运送到 Core B 以及将表型数据上传到每周一次。这将保证识别许多新的修饰突变，以促进活性项目 1 和项目 2。