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:
  • 金额:
    $ 62.42万
  • 依托单位:
  • 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

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 核心C的人员在饲养、安置和进行试验方面有多年的经验 NOD小鼠,在特定的无病原体条件下发生自发的T1D。一个非常具体的 对NOD小鼠的要求是无可挑剔的群体卫生,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都是如此。严格的同胞近亲繁殖程序,高度近亲繁殖的胚胎储存, 定期从这些种群中回收胚胎也是必要的,以防止导致 NOD/Nck H和NOD/Nck L亚系的建立。核心C将生成350个NOD/NKKH家系 经N-乙基-N-亚硝脲(ENU)修饰的G3小鼠诱发突变。在该计划期间产生的谱系 将显示平均约60个生殖系编码/剪接突变,其中包括80只G3雌性小鼠。 每只经过ENU处理的G0雄性将被培育成NOD/Nck H雌性,以获得10-20个G1雄性创建者。这些将是 回溯轨道放血以恢复高质量的DNA用于Illumina测序,并将使用尾部切割来分离 来自所有G2和G3女性的DNA用于离子激流测序进行基因分型;核心B将进行测序。 所有雌性G3幼崽将每隔一天监测一次T1D的发育,为期40天 几周。实验观察将包括首次检测到高血糖确认的葡萄糖尿症。为 所有小鼠的耳标号码、出生日期和表型数据都将被上传到Mutagenetix数据库,以便 卡普兰-迈耶分析可以每周进行一次,以考虑到每个谱系的新发展。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。初步数据证实了NOD/NKKH T1D修饰 可以实现表型,并立即绘制致病突变图谱,完全支持发现计划 我们建议。我们将进行长期监测(通过40周龄)来检测所有表型,包括 那些规模较小的突变,但我们将通过及早识别最强的致病突变来赢得时间。什么时候 每一只G1雄性已完成G2繁育,将采集附睾精子进行冷冻保存并公示 通过MMRRC分发;突变及其表型效应将在Mutagenetix上公布 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- 诱导等位基因。因此,如上所述,我们将把冷冻保存的精子从核心C转移到项目2,在那里 生殖系重定向将在NOD/Nck H株中发生。C核和B核之间的密切相互作用 将包括每月从核心C到核心B的DNA发货,以及在 每周一次。这将保证许多新的修饰突变的鉴定,以促进 项目1和项目2。

项目成果

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专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)

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Lucienne CHATENOUD其他文献

Lucienne CHATENOUD的其他文献

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{{ truncateString('Lucienne CHATENOUD', 18)}}的其他基金

Project 1 - Immunologic and Cellular Mechanisms of T1D Modifier Mutations
项目 1 - T1D 修饰突变的免疫学和细胞机制
  • 批准号:
    10642553
  • 财政年份:
    2023
  • 资助金额:
    $ 62.42万
  • 项目类别:

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