Chen Xiaochu, Xu Lan
Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Worcester, MA, USA.
Blueprint Medicines, 215 First Street, Cambridge, MA, USA.
Methods Mol Biol. 2016;1344:365-77. doi: 10.1007/978-1-4939-2966-5_24.
The transforming growth factor-β (TGF-β) family of cytokines figures prominently in regulation of embryonic development and adult tissue homeostasis from Drosophila to mammals. Genetic defects affecting TGF-β signaling underlie developmental disorders and diseases such as cancer in human. Therefore, delineating the molecular mechanism by which TGF-β regulates cell biology is critical for understanding normal biology and disease mechanisms. Forward genetic screens in model organisms and biochemical approaches in mammalian tissue culture were instrumental in initial characterization of the TGF-β signal transduction pathway. With complete sequence information of the genomes and the advent of RNA interference (RNAi) technology, genome-wide RNAi screening emerged as a powerful functional genomics approach to systematically delineate molecular components of signal transduction pathways. Here, we describe a protocol for image-based whole-genome RNAi screening aimed at identifying molecules required for TGF-β signaling into the nucleus. Using this protocol we examined >90 % of annotated Drosophila open reading frames (ORF) individually and successfully uncovered several novel factors serving critical roles in the TGF-β pathway. Thus cell-based high-throughput functional genomics can uncover new mechanistic insights on signaling pathways beyond what the classical genetics had revealed.
细胞因子转化生长因子-β（TGF-β）家族在从果蝇到哺乳动物的胚胎发育和成年组织稳态调节中起着重要作用。影响TGF-β信号传导的遗传缺陷是人类发育障碍和诸如癌症等疾病的基础。因此，阐明TGF-β调节细胞生物学的分子机制对于理解正常生物学和疾病机制至关重要。模式生物中的正向遗传筛选和哺乳动物组织培养中的生化方法有助于TGF-β信号转导途径的初步表征。随着基因组完整序列信息的获得以及RNA干扰（RNAi）技术的出现，全基因组RNAi筛选成为一种强大的功能基因组学方法，用于系统地描绘信号转导途径的分子成分。在这里，我们描述了一种基于图像的全基因组RNAi筛选方案，旨在鉴定TGF-β信号传入细胞核所需的分子。使用该方案，我们分别检查了>90%的注释果蝇开放阅读框（ORF），并成功发现了几个在TGF-β途径中起关键作用的新因子。因此，基于细胞的高通量功能基因组学可以揭示信号通路的新机制见解，这是经典遗传学所无法揭示的。
