Cell-based RNAi screening and high-content analysis in primary calvarian osteoblasts applied to identification of osteoblast differentiation regulators.
Cell-based RNAi screening and high-content analysis in primary calvarian osteoblasts applied to identification of osteoblast differentiation regulators.
复制标题
DOI：
10.1038/s41598-018-32364-8
复制
发表时间：
2018-09-19
影响因子：
4.6
通讯作者：
Tuckermann J
中科院分区：
文献类型：
--
作者：
Ahmad M;Kroll T;Jakob J;Rauch A;Ploubidou A;Tuckermann J
成骨细胞负责维持骨稳态。它们分化的失调在病因学上与几种骨疾病有关，使这一过程成为治疗干预的重要靶点。成骨细胞调节剂的系统性鉴定受到生理学相关的体外系统的不可用性的阻碍，该体外系统适用于有效的RNAi和与基于荧光显微镜的高含量分析（HCA）相容的分化读出。在这里，我们报告了一种新的方法，用于识别成骨细胞分化调节剂相结合的siRNA转染在生理相关的细胞与高通量筛选（HTS）。将原代小鼠颅骨成骨细胞接种在384孔板中，并用siRNA反向转染，并通过HCA测定其细胞数量和分化。自动图像采集允许高通量分析和单细胞特征分类。使用已知的成骨细胞分化调节剂验证了该方法的生理相关性、再现性和灵敏度。HCA对320个基因表达的siRNA的应用导致了对早期成骨细胞分化的5个潜在抑制因子和60个激活因子的鉴定。所描述的方法和相关的分析管道不限于基于RNAi的筛选，而是可以适用于大规模药物HTS或小规模靶向实验，以鉴定对早期成骨细胞生成重要的新的关键因素。
Osteoblasts are responsible for the maintenance of bone homeostasis. Deregulation of their differentiation is etiologically linked to several bone disorders, making this process an important target for therapeutic intervention. Systemic identification of osteoblast regulators has been hampered by the unavailability of physiologically relevant in vitro systems suitable for efficient RNAi and for differentiation read-outs compatible with fluorescent microscopy-based high-content analysis (HCA). Here, we report a new method for identification of osteoblast differentiation regulators by combining siRNA transfection in physiologically relevant cells with high-throughput screening (HTS). Primary mouse calvarial osteoblasts were seeded in 384-well format and reverse transfected with siRNAs and their cell number and differentiation was assayed by HCA. Automated image acquisition allowed high-throughput analyses and classification of single cell features. The physiological relevance, reproducibility, and sensitivity of the method were validated using known regulators of osteoblast differentiation. The application of HCA to siRNAs against expression of 320 genes led to the identification of five potential suppressors and 60 activators of early osteoblast differentiation. The described method and the associated analysis pipeline are not restricted to RNAi-based screening, but can be adapted to large-scale drug HTS or to small-scale targeted experiments, to identify new critical factors important for early osteoblastogenesis.
登录
查看更多内容
SRC抑制剂dasatinib加速了人骨骨髓衍生的间充质基质细胞分化成成骨细胞。
影响因子：
3.8
作者：
Id Boufker H;Lagneaux L;Najar M;Piccart M;Ghanem G;Body JJ;Journé F
通讯作者：
Journé F
Osf2/Cbfa1：成骨细胞分化的转录激活因子
影响因子：
64.5
作者：
Ducy, P;Zhang, R;Karsenty, G
通讯作者：
Karsenty, G
影响因子：
4.8
作者：
Americo-Da-Silva, Luan;Diaz, Jheimmy;Quiroga, Clara
通讯作者：
Quiroga, Clara
影响因子：
4.8
作者：
Liu, CJ;Chang, E;Di Cesare, PE
通讯作者：
Di Cesare, PE
影响因子：
8
作者：
Lee, Y-C;Huang, C-F;Murshed, M.;Chu, K.;Araujo, J. C.;Ye, X.;deCrombrugghe, B.;Yu-Lee, L-Y;Gallick, G. E.;Lin, S-H
通讯作者：
Lin, S-H