Genome-wide ENU mutagenesis screen for Runx2 modifiers

Runx2 修饰符的全基因组 ENU 诱变筛选

• 批准号: 6956298
• 负责人: Patricia Florence Ducy
• 金额: $ 9.75万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6956298
• 关键词: X ray; bioassay; biotechnology; bone development; bone development disorder; cell differentiation; developmental genetics; gene mutation; genetic mapping; genetic screening; laboratory mouse; mesenchyme; molecular cloning; nitrosourea; osteoblasts; phenotype; structural genes; transcription factor

DESCRIPTION (provided by applicant): The long-term goal of our project is to identify factors regulating mesenchymal cells commitment and early maturation into differentiated osteoblasts. As a first step toward this goal, we propose to perform a genome-wide genetic screen in mice using ethyl-nitrosourea (ENU) mutagenesis to expose mutations able to functionally compensate for Runx2 haploinsufficiency. Runx2 is the earliest known determinant of osteoblast differentiation, and loss of one Runx2 allele in mice or human causes Cleidocranial dysplasia (CCD), a skeletal disorder marked by clavicle agenesis and ossification defects. Any regulator of Runx2 expression or activity is thus likely to be involved in controlling either osteoblast differentiation or osteoblast function. Our strategy relies on a search for ENU-mutated animals that harbor a rescued CCD phenotype despite a Runx2 genetic background. We hypothesize that such rescuing mutations will affect regulators of Runx2 gene expression or protein stability/availability/activity or trigger yet unknown parallel or downstream compensating pathways. Subsequent characterization of the genes harboring these mutations should thus identify regulators involved in the early steps of skeletogenesis. The present application proposes to identify rescued Runx2 ENU mutants and to select the best candidates for subsequent mutation mapping and gene identification. Our specific aims are: to identify ENU-mutated mouse strains harboring a rescue of the CCD phenotype despite heterozygocity at the Runx2 locus; and to compare the mutant lines isolated and prioritize them for subsequent mutation mapping.

描述(由申请人提供)：我们项目的长期目标是确定调节间充质细胞承诺和分化成骨细胞的早期成熟的因素。作为实现这一目标的第一步，我们建议使用乙基亚硝胺(ENU)突变对小鼠进行全基因组遗传筛查，以揭示能够在功能上补偿Runx2单倍体不足的突变。Runx2是已知的最早的成骨细胞分化决定因素，在小鼠或人类中，Runx2等位基因缺失会导致锁骨颅骨发育不良(CCD)，这是一种以锁骨发育不全和骨化缺陷为特征的骨骼疾病。因此，Runx2表达或活性的任何调节因子都可能参与控制成骨细胞分化或成骨细胞功能。我们的策略依赖于寻找ENU突变的动物，这些动物具有挽救的CD表型，尽管有Runx2的遗传背景。我们推测，这种救助性突变将影响Runx2基因表达或蛋白质稳定性/可用性/活性的调节，或触发未知的平行或下游补偿途径。因此，随后对含有这些突变的基因的特征应该确定与骨骼形成的早期步骤有关的调节因子。本申请建议鉴定挽救的Runx2 ENU突变体，并为随后的突变作图和基因鉴定选择最佳候选者。我们的具体目标是：鉴定ENU突变的小鼠品系，尽管Runx2基因座为杂合子，但仍具有挽救Ccd表型的能力；并比较分离的突变株，并将它们优先用于后续的突变图谱。