Gromycin selection through use of a surrogate reporter system MedChemExpress AKT inhibitor 2 enriches nuclease-induced mutant cells. (A) Enrichment of GFP+ cells after hygromycin selection. Scale bar = 50 mm. (B) ZFN-driven mutations detected by the T7E1 assay. Arrows indicate the expected positions of DNA bands cleaved by mismatch-sensitive T7E1. The numbers at the bottom of the gel indicate mutation percentages calculated by band intensities. (C) DNA sequences of the wild-type (WT) and mutant clones, with ZFN recognition sites underlined. Dashes indicate deleted bases, and small bold letters indicate inserted bases. The number of occurrences is shown in parentheses; X1, X2, and X3 indicate the number of times that each clone was detected. Mutation ML-240 site frequencies were calculated by dividing the number of mutant clones by the number of total clones. doi:10.1371/journal.pone.0056476.gvia these new reporter systems was as efficient as that obtained via flow cytometry. We summarized the characteristics of the three reporter systems (Table 2). Hygromycin selection does not need any special instruments or machines, whereas flow cytometric sorting requires flow cytometers, which can be expensive and complicated. Magnetic separation requires magnetic separation instruments, which are much less expensive and simpler than flow cytometers. Thus, if these special facilities or instruments are not available, hygromycin selection would be the choice. If the time required for the enrichment process needs to be short, flow cytometric and magnetic separation would be preferred. These methods take only several hours, whereas hygromycin selection takes several days. Furthermore, hygromycin concentration and exposure time often needs to be determined for each cell type, whereas vigorous optimization processes are less critical in flow cytometric sorting and magnetic separation (although the performance of a flow cytometer machine should be optimized for proper cell sorting). If cells are sensitive to hydrostatic pressure and laser exposure, magnetic separation and hygromycin selection should be considered. Research environments vary and researchers can choose appropriate reporters depending on their experimental conditions.In addition, the magnetic and hygromycin reporters can be also used for flow cytometric enrichment of mutant cells because these two reporters express GFP in addition to H-2Kk or HygroR when indels are generated in their target sequences. Thus, our two new reporters will practically replace the previously described fluorescent reporters.ConclusionsHere we described two novel episomal reporter systems that can enrich cells with nuclease-induced mutations using magnetic separation and hygromycin selection. The magnetic and hygromycin 15900046 reporters contain the target sequences of the engineered nucleases and express H-2Kk and HygroR, respectively, only when indels are generated in the target sequences by the activity of engineered nucleases. The mutant cell enrichment efficiencies using magnetic and hygromycin reporters were comparable to that using the previously reported fluorescent reporters. Furthermore, our new reporters also allow mutant cell enrichment using flow cytometers as well. Given that ZFNs and TALENs are used in various research environments, our two new reporters will practically replace the previously reported fluorescent reporterFlow Cytometer-Free Enrichment of Mutant CellsFigure 6. Clonal analysis of hygromycin-selected colonies. After CCR5-targeting ZFN (.Gromycin selection through use of a surrogate reporter system enriches nuclease-induced mutant cells. (A) Enrichment of GFP+ cells after hygromycin selection. Scale bar = 50 mm. (B) ZFN-driven mutations detected by the T7E1 assay. Arrows indicate the expected positions of DNA bands cleaved by mismatch-sensitive T7E1. The numbers at the bottom of the gel indicate mutation percentages calculated by band intensities. (C) DNA sequences of the wild-type (WT) and mutant clones, with ZFN recognition sites underlined. Dashes indicate deleted bases, and small bold letters indicate inserted bases. The number of occurrences is shown in parentheses; X1, X2, and X3 indicate the number of times that each clone was detected. Mutation frequencies were calculated by dividing the number of mutant clones by the number of total clones. doi:10.1371/journal.pone.0056476.gvia these new reporter systems was as efficient as that obtained via flow cytometry. We summarized the characteristics of the three reporter systems (Table 2). Hygromycin selection does not need any special instruments or machines, whereas flow cytometric sorting requires flow cytometers, which can be expensive and complicated. Magnetic separation requires magnetic separation instruments, which are much less expensive and simpler than flow cytometers. Thus, if these special facilities or instruments are not available, hygromycin selection would be the choice. If the time required for the enrichment process needs to be short, flow cytometric and magnetic separation would be preferred. These methods take only several hours, whereas hygromycin selection takes several days. Furthermore, hygromycin concentration and exposure time often needs to be determined for each cell type, whereas vigorous optimization processes are less critical in flow cytometric sorting and magnetic separation (although the performance of a flow cytometer machine should be optimized for proper cell sorting). If cells are sensitive to hydrostatic pressure and laser exposure, magnetic separation and hygromycin selection should be considered. Research environments vary and researchers can choose appropriate reporters depending on their experimental conditions.In addition, the magnetic and hygromycin reporters can be also used for flow cytometric enrichment of mutant cells because these two reporters express GFP in addition to H-2Kk or HygroR when indels are generated in their target sequences. Thus, our two new reporters will practically replace the previously described fluorescent reporters.ConclusionsHere we described two novel episomal reporter systems that can enrich cells with nuclease-induced mutations using magnetic separation and hygromycin selection. The magnetic and hygromycin 15900046 reporters contain the target sequences of the engineered nucleases and express H-2Kk and HygroR, respectively, only when indels are generated in the target sequences by the activity of engineered nucleases. The mutant cell enrichment efficiencies using magnetic and hygromycin reporters were comparable to that using the previously reported fluorescent reporters. Furthermore, our new reporters also allow mutant cell enrichment using flow cytometers as well. Given that ZFNs and TALENs are used in various research environments, our two new reporters will practically replace the previously reported fluorescent reporterFlow Cytometer-Free Enrichment of Mutant CellsFigure 6. Clonal analysis of hygromycin-selected colonies. After CCR5-targeting ZFN (.
