Core C - Mutagenesis, Screening and Cryopreservation

核心 C - 诱变、筛选和冷冻保存

• 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
• 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

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.

项目总结/摘要 核心C的工作人员在繁殖、圈养和进行实验方面有多年的经验， NOD小鼠，其在无特定病原体的条件下发生自发性T1 D。一个非常具体 对NOD小鼠的要求是殖民地无可挑剔的卫生，因为最小的变化可能会影响T1 D的发病率 在雌性和雄性小鼠中。一个严格的近亲繁殖程序，从高度近亲繁殖的种群中储存胚胎， 定期从这一种群中回收胚胎也是必要的，以防止导致遗传漂变的类型。 建立NOD/NckH和NOD/NckL亚系。核心C将产生350个NOD/NckH谱系 通过N-乙基-N亚硝基脲（ENU）诱导的突变修饰的G3小鼠。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只G3雌性小鼠。 将每只ENU处理的G 0雄性与NOD/NckH雌性交配，以获得10-20只G1雄性建立者。这些将是 逆轨道流血的以回收用于Illumina测序的高质量DNA，并且尾切将用于分离 所有G2和G3雌性动物的DNA用于通过Ion Torrent测序进行基因分型;核心B将进行测序。 在40天的时间内，每隔一天监测所有雌性G3幼仔的T1 D发展情况 周实验观察将包括首次检测糖尿，并通过高血糖症证实。为 所有小鼠耳标编号、出生日期和表型数据将上传至Mutagenetix数据库，以便 Kaplan-Meier分析可以每周进行一次，以考虑每个谱系中的新发展。我们 将对携带CRISPR/Cas9靶向突变的家系进行类似的表型分析， 项目2，用于核查目的。初步数据证实，NOD/NckH T1 D的修改 表型可以实现，致病突变立即映射，完全支持发现计划 我们提议。我们将进行延长监测（至40周龄），以检测所有表型，包括 那些较小的幅度，但我们将赢得时间，确定最强的致病突变早期。 每只G1代雄性完成G2代交配后，将收获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 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-1基因反式放置。 诱导等位基因因此，如前所述，我们将把冷冻保存的精子从核心C转移到项目2， 在NOD/NckH菌株中将发生种系再靶向。核心C和核心B之间的密切互动 将包括每月从核心C向核心B运送DNA，并在 每周的基础上。这将保证许多新的修饰突变的鉴定，以促进活性。 项目1和项目2。