Genome-wide ENU mutagenesis screen for Runx2 modifiers

项目描述（由申请人提供）：本项目的长期目标是确定调节间充质细胞承诺和早期成熟为分化成骨细胞的因素。作为实现这一目标的第一步，我们建议在小鼠中使用乙基亚硝基脲（ENU）诱变进行全基因组遗传筛选，以暴露能够在功能上补偿Runx2单倍不足的突变。Runx2是已知最早的成骨细胞分化的决定因素，在小鼠或人类中丢失一个Runx2等位基因会导致锁骨发育不良（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，这些动物尽管具有Runx2遗传背景，但仍具有获救的CCD表型。我们假设这种挽救性突变将影响Runx2基因表达或蛋白质稳定性/可用性/活性的调节因子，或触发未知的平行或下游补偿途径。随后对包含这些突变的基因进行表征，从而确定参与骨骼形成早期步骤的调节因子。本应用程序旨在鉴定获救的Runx2 ENU突变体，并为随后的突变定位和基因鉴定选择最佳候选体。我们的具体目标是：鉴定enu突变的小鼠菌株，尽管在Runx2位点存在杂合子，但仍具有CCD表型的拯救；并比较分离的突变系，为后续的突变定位确定优先级。

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.