Skeletal Phenotyping of Heterozygotes from IMPC Embryonic Lethal Lines

摘要 国际老鼠表型鉴定联盟(IMPC)开发了一个无价的基因库 基因敲除(KO)小鼠，并对每个KO系进行了整体动物评估。大约1/3的人 KO系是以杂合子(HET)KO系为特征的胚胎致死系。以前我们 对220例未经选择的存活纯合子(HOM)进行高通量µCT和组织形态计量学分析 Ko Lines。这项研究发现，~12.5%的人在总骨量(骨小梁)方面存在明确的差异 体积和/或骨大小)，其中大部分未被IMPC筛查发现。结果和 屏幕上检查的KO的解释可在门户网站(bonebase.org)上找到。在此基础上 经验，IMPC门户网站上的表型数据和文献，我们估计HET KO群体将是影响骨/身体差异和骨骼健康的同样丰富的基因来源 成年后的岁月。这项建议将我们的骨架表型工作流程应用于选定的HET KO品系，这些品系 可能具有显著的骨/体质量表型。微CT和全身成分均为 经TD-核磁共振测定，将作为筛选方式。骨变异显著的HET KO系 将对建筑进行组织形态计量学处理。基于这些结果，某些线路将进行研究 成骨(矿物/基质成分的拉曼显微镜，成骨细胞的原代细胞培养) 和/或破骨细胞生成(胶原交联物和破骨细胞原代培养)。在HET KO线路中 将进行身体成分的变化，骨骼肌的组织形态计量学。其目的是为了 区分HET KO的影响是直接作用于成骨细胞还是破骨细胞，或者是否 观察到的表型是HET KO对其他组织的继发性作用，可以影响骨骼。此外, 存活到妊娠18.5天的Hom KO致死株将进行大体和组织学骨骼观察 检查以确定基因对骨骼发育的影响。以补充这些生物 发现，正在添加一个生物信息学组件来绘制影响成骨细胞或 破骨细胞直接通向已知或假定的分子途径。该基因是否参与了 网络还是网络的产物？该项目的最终目标是将生物和生物信息学 将数据转换为分类结构，以显示不同KO行之间的差异和相似之处。我们 将聘请骨生物学的内部和外部专家来审查为特定HET KO收集的数据 线，以批评和扩大我们的解释和不断演变的分类模式。认识到 影响骨骼的基因的频率和复杂性是一个大数据挑战(估计有~3500个基因)， 我们的目的是为如何收集、呈现、解释和查询这些信息奠定基础 并最终应用于个性化骨骼人类健康的目标。

Abstract The International Mouse Phenotyping Consortium (IMPC) has developed an invaluable repository of gene knockout (KO) mice and has performed a whole-animal assessment of each KO line. Approximately 1/3 of the KO lines are embryonic lethals that were characterized as heterozygous (HET) KO lines. Previously we performed high throughput µCT and histomorphometric analysis on 220 unselected viable homozygous (HOM) KO lines. This study found that ~12.5% have an unequivocal variance in total bone mass (trabecular bone volume and/or bone size), most of which were not detected by the IMPC screening. The results and interpretation of the KOs examined in the screen are available on a web portal (bonebase.org). Based on this experience, the phenotyping data on the IMPC webportal and the literature, we estimate that the HET population of KOs will be an equally rich source of genes that affect bone/body variance and skeletal health in the later adult years. This proposal will apply our skeletal phenotyping workflow to selected HET KO lines that are likely to have a significant bone/body mass phenotype. Both µCT and total body composition as determined by TD-NMR will be used as the screening modality. HET KO lines with significant variance in bone architecture will be processed for histomorphometry. Based on those results, certain lines will undergo studies of osteogenesis (Raman microscopy for the mineral/matrix composition, primary cell culture for osteoblast) and/or osteoclastogenesis (collagen crosslinks and primary cell culture for osteoclast). In HET KO lines with variance in body composition, histomorphometry of the skeletal muscle will be performed. The intent is to discriminate the impact of the HET KO as acting directly on the osteoblast or osteoclast, or whether the observed phenotype is a secondary effect of the HET KO on other tissues that can influence bone. In addition, HOM KO lethal lines that survive to 18.5 days of gestation will undergo a gross and histological skeletal examination to determine the impact of the gene on skeletal development. To complement these biological findings, a bioinformatics component is being added to map the KO genes that are affecting osteoblast or osteoclast directly to known or postulated molecular pathways. Does the gene participate in the function of the network or is it a product of a network? The final goal of the project is to place the biological and bioinformatic data into a classification structure that reveals differences and similarities between the various KO lines. We will engage internal and external experts in bone biology to review the data assembled for a specific HET KO line to critique and amplify our interpretation and ever-evolving classification schema. Recognizing that frequency and complexity of the genes affecting bone is a big-data challenge (estimated to be ~3,500 genes), our intent is to lay the ground work for how this information will be gathered, presented, interpreted, queried and eventually applied toward the goal of personalized skeletal human health.