Analyzing Homologous Recombination Using Antibiotic Marker Substrates in Mammalian Cells

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.