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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.

项目总结/文摘