Badakul Gamze, Nickoloff Jac A, Kato Takamitsu A
Department of Environmental & Radiological Health Sciences, Colorado State University, Fort Collins, CO, USA.
Methods Mol Biol. 2025;2933:141-148. doi: 10.1007/978-1-0716-4574-1_18.
Mammalian cell lines with single-copy, integrated plasmid homologous recombination (HR) substrates are valuable tools to assess spontaneous and DNA double-strand break (DSB)-induced HR in vivo. Two common systems employ repeats of fluorescent markers (e.g., GFP) or antibiotic-resistance markers (e.g., neo). Here, we describe the use of Chinese hamster ovary (CHO) strain 33 as an effective model system for studying DSB-induced HR, a critical DSB repair pathway that generally preserves genomic integrity. This assay utilizes the I-SceI endonuclease from Saccharomyces cerevisiae, which recognizes a unique 18 bp sequence to induce a targeted DSB, thereby triggering HR between neo direct repeats. In this system, one neo allele, referred to as neo12, contains silent mutations that reduce spontaneous recombination, while the other allele is inactivated by an I-SceI restriction site, allowing regulated initiation of recombination. CHO strain 33 demonstrates an exceptionally low background recombination rate in comparison to the direct repeat green fluorescent protein (DR-GFP) assay, which has high spontaneous activity. Furthermore, recombination frequency in CHO strain 33 can be quantified precisely using colony formation assays, eliminating the need for microscopy or flow cytometry, which is typically required to analyze HR with DR-GFP. This protocol outlines comprehensive methods for detecting I-SceI-induced neo gene conversion or single-strand annealing (SSA) in CHO strain 33. This general protocol can be extended to other cell lines with wild-type or mutant DNA repair and DNA damage checkpoint genetic backgrounds, providing a robust framework for understanding HR mechanisms and evaluating the impact of DNA repair and checkpoint defects on DSB-induced HR repair frequencies and HR product spectra.
具有单拷贝整合质粒同源重组（HR）底物的哺乳动物细胞系是评估体内自发和DNA双链断裂（DSB）诱导的HR的重要工具。两种常见系统采用荧光标记（如绿色荧光蛋白，GFP）或抗生素抗性标记（如新霉素抗性基因，neo）的重复序列。在此，我们描述了使用中国仓鼠卵巢（CHO）33株作为研究DSB诱导的HR的有效模型系统，DSB诱导的HR是一种关键的DSB修复途径，通常可维持基因组完整性。该检测利用来自酿酒酵母的I-SceI内切酶，它识别一个独特的18bp序列以诱导靶向DSB，从而触发neo直接重复序列之间的HR。在这个系统中，一个neo等位基因，称为neo12，含有沉默突变，可降低自发重组，而另一个等位基因则被I-SceI限制位点灭活，从而允许调控重组的起始。与具有高自发活性的直接重复绿色荧光蛋白（DR-GFP）检测相比，CHO 33株表现出极低的背景重组率。此外，CHO 33株中的重组频率可以通过集落形成试验精确量化，无需使用显微镜或流式细胞术，而分析DR-GFP的HR通常需要这些技术。本方案概述了检测CHO 33株中I-SceI诱导的neo基因转换或单链退火（SSA）的综合方法。这个通用方案可以扩展到具有野生型或突变DNA修复和DNA损伤检查点遗传背景的其他细胞系，为理解HR机制以及评估DNA修复和检查点缺陷对DSB诱导的HR修复频率和HR产物谱的影响提供了一个强大的框架。
