D.Flow Cytometer-Free Enrichment of Mutant CellsFigure 1. Overview of the episomal Title Loaded From File reporters used for the enrichment of nuclease-induced mutant cells via magnetic separation. (A) The working mechanism of the H-2Kk magnetic reporter. mRFP is constitutively expressed by the CMV promoter (PCMV), whereas eGFP and H-2Kk are not expressed without the activity of engineered nucleases because their sequences are out of frame. If a double-strand break is introduced into the target sequence by engineered nucleases, the break is repaired by nonhomologous end-joining (NHEJ), which often results in indels. Indel generation can cause frame shifts, making eGFP and H-2Kk in frame and leading to the expression of eGFP and H-2Kk. (B) A schematic depicting the enrichment of mutant cells using the H-2Kk reporter. H-2Kk-expressing cells can be magnetically separated using anti-H-2kk antibody conjugated to magnetic beads. Mutant cells were enriched in this population of H-2kk-expressing cells. Reporter plasmids and chromosomal target loci are shown. Black spots represent mutations. doi:10.1371/journal.pone.0056476.gHere we present two novel reporter systems that enable enrichment of nuclease-induced mutant cells using magnetic separation and hygromycin selection. These reporters express H2Kk and the hygromycin resistance protein, respectively, only when insertions or deletions (indels) are generated at the target sequences in the reporter systems, enabling efficient enrichment of mutant cells without using a flow cytometer.ZFNs, TALENs, and reportersPlasmids encoding the ZFNs and TALENs used in this study were previously described [3,23] or obtained from ToolGen (Seoul, South Korea). Reporters were prepared as previously described [3]. Briefly, oligonucleotides that contained target sites were synthesized (Bioneer, Daejon, South Korea) and annealed in vitro. The annealed oligonucleotides were ligated into the vector. The sequences of reporters that contain Z891 target sites are included in Notes S1 and S2.Materials and Methods Reporter vector Title Loaded From File constructionThe 2A sequence was inserted into the pRGS reporter [3] using synthesized oligonucleotides (Bioneer, Daejon, South Korea). The mouse H2-Kk gene was amplified from pMACS Kk (Miltenyi 15857111 Biotech, Germany) using appropriate primers (Table S1), and the amplified product was cloned into the modified pRGS vector by isothermal cloning [22]. The hygromycin B resistance gene was amplified from pBABE-hygro-hTERT (Addgene, plasmid #1773) using appropriate primers (Table S1), and the amplified product was cloned into the NheI site of the modified pRGS vector.Cell cultureHuman embryonic kidney 293T (HEK293T) cells and Huh 7.5 cells were maintained in Dulbecco’s modified Eagle medium (DMEM, Invitrogen) supplemented with 100 units/ml penicillin, 100 mg/ml streptomycin, and 10 fetal bovine serum.TransfectionCells were transfected using lipofectamine 2000 (Invitrogen, Carlsbad, CA) or polyethyleneimine (linear, MW,25,000, Polysciences) at a weight ratio of 1:1:2 (plasmid encoding a ZFN: plasmid encoding the other ZFN: magnetic reporter) or 10:10:Flow Cytometer-Free Enrichment of Mutant CellsFigure 2. Magnetic separation-mediated enrichment of CCR5-disrupted cells using an episomal reporter. (A) Enrichment of GFP+ cells after magnetic separation. 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 botto.D.Flow Cytometer-Free Enrichment of Mutant CellsFigure 1. Overview of the episomal reporters used for the enrichment of nuclease-induced mutant cells via magnetic separation. (A) The working mechanism of the H-2Kk magnetic reporter. mRFP is constitutively expressed by the CMV promoter (PCMV), whereas eGFP and H-2Kk are not expressed without the activity of engineered nucleases because their sequences are out of frame. If a double-strand break is introduced into the target sequence by engineered nucleases, the break is repaired by nonhomologous end-joining (NHEJ), which often results in indels. Indel generation can cause frame shifts, making eGFP and H-2Kk in frame and leading to the expression of eGFP and H-2Kk. (B) A schematic depicting the enrichment of mutant cells using the H-2Kk reporter. H-2Kk-expressing cells can be magnetically separated using anti-H-2kk antibody conjugated to magnetic beads. Mutant cells were enriched in this population of H-2kk-expressing cells. Reporter plasmids and chromosomal target loci are shown. Black spots represent mutations. doi:10.1371/journal.pone.0056476.gHere we present two novel reporter systems that enable enrichment of nuclease-induced mutant cells using magnetic separation and hygromycin selection. These reporters express H2Kk and the hygromycin resistance protein, respectively, only when insertions or deletions (indels) are generated at the target sequences in the reporter systems, enabling efficient enrichment of mutant cells without using a flow cytometer.ZFNs, TALENs, and reportersPlasmids encoding the ZFNs and TALENs used in this study were previously described [3,23] or obtained from ToolGen (Seoul, South Korea). Reporters were prepared as previously described [3]. Briefly, oligonucleotides that contained target sites were synthesized (Bioneer, Daejon, South Korea) and annealed in vitro. The annealed oligonucleotides were ligated into the vector. The sequences of reporters that contain Z891 target sites are included in Notes S1 and S2.Materials and Methods Reporter vector constructionThe 2A sequence was inserted into the pRGS reporter [3] using synthesized oligonucleotides (Bioneer, Daejon, South Korea). The mouse H2-Kk gene was amplified from pMACS Kk (Miltenyi 15857111 Biotech, Germany) using appropriate primers (Table S1), and the amplified product was cloned into the modified pRGS vector by isothermal cloning [22]. The hygromycin B resistance gene was amplified from pBABE-hygro-hTERT (Addgene, plasmid #1773) using appropriate primers (Table S1), and the amplified product was cloned into the NheI site of the modified pRGS vector.Cell cultureHuman embryonic kidney 293T (HEK293T) cells and Huh 7.5 cells were maintained in Dulbecco’s modified Eagle medium (DMEM, Invitrogen) supplemented with 100 units/ml penicillin, 100 mg/ml streptomycin, and 10 fetal bovine serum.TransfectionCells were transfected using lipofectamine 2000 (Invitrogen, Carlsbad, CA) or polyethyleneimine (linear, MW,25,000, Polysciences) at a weight ratio of 1:1:2 (plasmid encoding a ZFN: plasmid encoding the other ZFN: magnetic reporter) or 10:10:Flow Cytometer-Free Enrichment of Mutant CellsFigure 2. Magnetic separation-mediated enrichment of CCR5-disrupted cells using an episomal reporter. (A) Enrichment of GFP+ cells after magnetic separation. 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 botto.

D.Flow Cytometer-Free Enrichment of Mutant CellsFigure 1. Overview of the episomal

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