Knowles Sara, Chai Weihang
Department of Cancer Biology, Cardinal Bernardin Cancer Center, Loyola University Chicago Stritch School of Medicine, Maywood, IL, USA.
Center for Genetic Diseases, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA.
Bio Protoc. 2024 Apr 20;14(8):e4977. doi: 10.21769/BioProtoc.4977.
The CTC1-STN1-TEN1 (CST) complex is a single-strand DNA-binding protein complex that plays an important role in genome maintenance in various model eukaryotes. Dysfunction of CST is the underlying cause of the rare genetic disorder known as Coats plus disease. In addition, down regulation of STN1 promotes colorectal cancer development in mice. While prior studies have utilized RNAi to knock down CST components in mammalian cells, this approach is associated with off-target effects. Attempts to employ CRISPR/Cas9-based knockout of CST components in somatic cell lines have been unsuccessful due to CST's indispensable role in DNA replication and cell proliferation. To address these challenges, we outline a novel approach utilizing a Cre-loxP-based conditional knockout in mouse embryonic fibroblasts (MEFs). This method offers an alternative means to investigate the function and characteristics of the CST complex in mammalian systems, potentially shedding new light on its roles in genome maintenance. Key features • Conditional depletion of mammalian STN1 using mouse embryonic fibroblast (MEFs). • Analysis of oxidative damage sensitivity using STN1-depleted MEFs. • This protocol requires mice.
CTC1-STN1-TEN1（CST）复合物是一种单链DNA结合蛋白复合物，在多种模式真核生物的基因组维持中发挥重要作用。CST功能障碍是罕见遗传病科茨加综合征的根本原因。此外，STN1的下调促进小鼠结直肠癌的发展。虽然先前的研究利用RNA干扰在哺乳动物细胞中敲低CST成分，但这种方法存在脱靶效应。由于CST在DNA复制和细胞增殖中不可或缺的作用，在体细胞系中采用基于CRISPR/Cas9的CST成分敲除的尝试均未成功。为应对这些挑战，我们概述了一种在小鼠胚胎成纤维细胞（MEF）中利用基于Cre-loxP的条件性敲除的新方法。该方法为研究CST复合物在哺乳动物系统中的功能和特性提供了一种替代手段，可能为其在基因组维持中的作用带来新的启示。关键特性• 使用小鼠胚胎成纤维细胞（MEF）对哺乳动物STN1进行条件性缺失。• 使用STN1缺失的MEF分析氧化损伤敏感性。• 本方案需要小鼠。
