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.

项目摘要/摘要 C核C的人员在育种，住房和进行实验方面具有多年的经验点头小鼠，在没有特定的无病原体条件下会自发T1D。一个非常具体的对小鼠的需求是菌落无可挑剔的卫生，因为最小的变化可能会影响T1D发病率在男性和雄性小鼠中。一个严格的兄弟姐妹近交计划，从高度近交股中存储胚胎，并且还需要定期从该股票中回收胚胎，以防止导致的类型的遗传漂移建立点头/NCKH和点头/NCKL sublines。核心C将产生350个nod/nckh的血统通过N-乙基-N硝酸（ENU）诱导的突变修饰的G3小鼠。程序期间产生的血统书平均显示约60种种系编码/剪接突变，包括80 G3雌性小鼠。每个经过ENU处理的G0雄性将被育成点头/NCKH女性，以获得10-20 G1男性创始人。这些将是逆转录曲线以恢复高质量的DNA进行Illumina测序，将使用尾片来隔离来自所有G2和G3雌性的DNA，用于通过离子洪流测序进行基因分型；核心B将执行测序。所有雌性G3幼崽将每隔一天进行一次监测，以在40个时期内开发T1D 几周。实验观察结果将包括通过高血糖证实的首次检测葡萄糖尿。为了所有小鼠耳号的数字，出生日期和表型数据都将上传到诱变的数据库，以便可以每周进行Kaplan-Meier分析，以考虑每个血统中的新发展。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，出于验证目的。初步数据证实了NOD/NCKH T1D的修改可以实现表型，并立即映射致病突变，完全支持发现程序我们建议。我们将进行扩展监测（至40周龄），以检测所有表型那些幅度较小的人，但我们将通过早期确定最强的因果突变来获得时间。什么时候每个G1雄性都已完成G2育种，将收获附睾精子进行冷冻保存和公众通过MMRRC分发；突变及其表型效应将在诱变型上公开网站。有时，因果关系的验证取决于将基因敲除等位基因与原始的eNu- 诱导等位基因。因此，如前所述，我们将将冷冻保存的精子从核心C转移到项目2，生殖线重新定位将在点头/NCKH菌株中进行。 Core C和Core B之间的紧密相互作用将包括以每月从核心C到核B的每月间隔运输DNA，并在A上上传表型数据每周。这将确保识别许多新型修饰符突变，以助长项目1和项目2。