Magnetic Separation and Antibiotics Selection Enable Enrichment of Cells with ZFN/TALEN-Induced Mutations
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{"title"=>"Magnetic Separation and Antibiotics Selection Enable Enrichment of Cells with ZFN/TALEN-Induced Mutations", "type"=>"journal", "authors"=>[{"first_name"=>"Hyojin", "last_name"=>"Kim", "scopus_author_id"=>"37021670500"}, {"first_name"=>"Myung Sun", "last_name"=>"Kim", "scopus_author_id"=>"55686390200"}, {"first_name"=>"Gabbine", "last_name"=>"Wee", "scopus_author_id"=>"55603534700"}, {"first_name"=>"Choong Il", "last_name"=>"Lee", "scopus_author_id"=>"55603503000"}, {"first_name"=>"Hyongbum", "last_name"=>"Kim", "scopus_author_id"=>"24280276300"}, {"first_name"=>"Jin Soo", "last_name"=>"Kim", "scopus_author_id"=>"55949820500"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "scopus"=>"2-s2.0-84874185862", "sgr"=>"84874185862", "pui"=>"368381720", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "pmid"=>"23441197", "doi"=>"10.1371/journal.pone.0056476"}, "id"=>"0ee54dda-e7c4-3e1d-ac74-d2ad84f684ed", "abstract"=>"The ability to enrich cells with targeted mutations greatly facilitates the process of using engineered nucleases, including zinc-finger nucleases and transcription activator-like effector nucleases, to construct such cells. We previously used surrogate reporters to enrich cells containing nuclease-induced mutations via flow cytometry. This method is, however, limited by the availability of flow cytometers. Furthermore, sorted cells occasionally fail to form colonies after exposure to a strong laser and hydrostatic pressure. Here we describe two different types of novel reporters that enable mutant cell enrichment without the use of flow cytometers. We designed reporters that express H-2K(k), a surface antigen, and the hygromycin resistance protein (Hygro(R)), respectively, when insertions or deletions are generated at the target sequences by the activity of engineered nucleases. After cotransfection of these reporters and the engineered nuclease-encoding plasmids, H-2K(k)- and Hygro(R)-expressing cells were isolated using magnetic separation and hygromycin treatment, respectively. We found that mutant cells were drastically enriched in the isolated cells, suggesting that these two reporters enable efficient enrichment of mutants. We propose that these two reporters will greatly facilitate the use of engineered nucleases in a wider range of biomedical research.", "link"=>"http://www.mendeley.com/research/magnetic-separation-antibiotics-selection-enable-enrichment-cells-zfntaleninduced-mutations", "reader_count"=>98, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Professor > Associate Professor"=>6, "Researcher"=>23, "Student > Doctoral Student"=>4, "Student > Ph. D. Student"=>34, "Student > Postgraduate"=>1, "Student > Master"=>17, "Other"=>4, "Student > Bachelor"=>3, "Professor"=>5}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Professor > Associate Professor"=>6, "Researcher"=>23, "Student > Doctoral Student"=>4, "Student > Ph. D. Student"=>34, "Student > Postgraduate"=>1, "Student > Master"=>17, "Other"=>4, "Student > Bachelor"=>3, "Professor"=>5}, "reader_count_by_subject_area"=>{"Unspecified"=>2, "Biochemistry, Genetics and Molecular Biology"=>16, "Agricultural and Biological Sciences"=>69, "Medicine and Dentistry"=>5, "Neuroscience"=>2, "Veterinary Science and Veterinary Medicine"=>1, "Immunology and Microbiology"=>3}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>5}, "Neuroscience"=>{"Neuroscience"=>2}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>3}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>69}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>16}, "Unspecified"=>{"Unspecified"=>2}, "Veterinary Science and Veterinary Medicine"=>{"Veterinary Science and Veterinary Medicine"=>1}}, "reader_count_by_country"=>{"Turkey"=>1, "United States"=>4, "Japan"=>1, "Italy"=>1, "United Kingdom"=>1, "France"=>3}, "group_count"=>3}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/486898"], "description"=>"<p>(A) The working mechanism of the H-2K<sup>k</sup> magnetic reporter. mRFP is constitutively expressed by the CMV promoter (P<sub>CMV</sub>), whereas eGFP and H-2K<sup>k</sup> 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-2K<sup>k</sup> in frame and leading to the expression of eGFP and H-2K<sup>k</sup>. (B) A schematic depicting the enrichment of mutant cells using the H-2K<sup>k</sup> reporter. H-2K<sup>k</sup>-expressing cells can be magnetically separated using anti-H-2k<sup>k</sup> antibody conjugated to magnetic beads. Mutant cells were enriched in this population of H-2k<sup>k</sup>-expressing cells. Reporter plasmids and chromosomal target loci are shown. Black spots represent mutations.</p>", "links"=>[], "tags"=>["episomal", "reporters", "enrichment", "nuclease-induced", "mutant", "cells"], "article_id"=>157409, "categories"=>["Biotechnology", "Biochemistry", "Genetics", "Biological Sciences"], "users"=>["Hyojin Kim", "Myung-Sun Kim", "Gabbine Wee", "Choong-il Lee", "Hyongbum Kim", "Jin-Soo Kim"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0056476.g001", "stats"=>{"downloads"=>1, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Overview_of_the_episomal_reporters_used_for_the_enrichment_of_nuclease_induced_mutant_cells_via_magnetic_separation_/157409", "title"=>"Overview of the episomal reporters used for the enrichment of nuclease-induced mutant cells via magnetic separation.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-18 02:03:29"}
  • {"files"=>["https://ndownloader.figshare.com/files/487305"], "description"=>"<p>(A) Enrichment of GFP<sup>+</sup> cells after hygromycin selection. Scale bar = 50 µm. (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.</p>", "links"=>[], "tags"=>["surrogate", "enriches", "nuclease-induced", "mutant"], "article_id"=>157826, "categories"=>["Biotechnology", "Biochemistry", "Genetics", "Biological Sciences"], "users"=>["Hyojin Kim", "Myung-Sun Kim", "Gabbine Wee", "Choong-il Lee", "Hyongbum Kim", "Jin-Soo Kim"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0056476.g005", "stats"=>{"downloads"=>0, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Hygromycin_selection_through_use_of_a_surrogate_reporter_system_enriches_nuclease_induced_mutant_cells_/157826", "title"=>"Hygromycin selection through use of a surrogate reporter system enriches nuclease-induced mutant cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-18 02:10:26"}
  • {"files"=>["https://ndownloader.figshare.com/files/487188"], "description"=>"<p>(A) The working mechanism of the hygromycin reporter. mRFP is constitutively expressed by the CMV promoter (P<sub>CMV</sub>), whereas the <i>Hygro<sup>R</sup>-eGFP</i> fusion gene is not expressed in the absence of engineered nucleases because the <i>Hygro<sup>R</sup></i> and <i>eGFP</i> sequences are out of frame. If a double-strand break is introduced into the target sequence by engineered nucleases, the break is repaired by non-homologous end-joining (NHEJ), which often results in indels. Indel generation can cause frame shifts, rendering <i>Hygro<sup>R</sup>-eGFP</i> in frame and expressed. (B) A schematic depicting the enrichment of mutant cells using the hygromycin reporter. <i>Hygro<sup>R</sup>-eGFP</i> fusion gene-expressing cells can be selected using hygromycin treatment. Mutant cells were enriched in this population of <i>Hygro<sup>R</sup>-eGFP</i>-expressing cells. Reporter plasmids and chromosomal target loci are shown. Black spots represent mutations.</p>", "links"=>[], "tags"=>["episomal", "reporters", "enrichment", "nuclease-induced", "mutant", "cells", "hygromycin"], "article_id"=>157699, "categories"=>["Biotechnology", "Biochemistry", "Genetics", "Biological Sciences"], "users"=>["Hyojin Kim", "Myung-Sun Kim", "Gabbine Wee", "Choong-il Lee", "Hyongbum Kim", "Jin-Soo Kim"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0056476.g004", "stats"=>{"downloads"=>1, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Overview_of_the_episomal_reporters_used_for_the_enrichment_of_nuclease_induced_mutant_cells_via_hygromycin_selection_/157699", "title"=>"Overview of the episomal reporters used for the enrichment of nuclease-induced mutant cells via hygromycin selection.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-18 02:08:19"}
  • {"files"=>["https://ndownloader.figshare.com/files/1004664"], "description"=>"<p>Comparison of enrichment methods.</p>", "links"=>[], "tags"=>["enrichment"], "article_id"=>665287, "categories"=>["Biotechnology", "Biochemistry", "Genetics", "Biological Sciences"], "users"=>["Hyojin Kim", "Myung-Sun Kim", "Gabbine Wee", "Choong-il Lee", "Hyongbum Kim", "Jin-Soo Kim"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0056476.t002", "stats"=>{"downloads"=>8, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Comparison_of_enrichment_methods_/665287", "title"=>"Comparison of enrichment methods.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-02-18 01:28:07"}
  • {"files"=>["https://ndownloader.figshare.com/files/1004645"], "description"=>"<p>Efficiencies of mutant cell enrichment via different reporter systems.</p>", "links"=>[], "tags"=>["mutant", "enrichment"], "article_id"=>665267, "categories"=>["Biotechnology", "Biochemistry", "Genetics", "Biological Sciences"], "users"=>["Hyojin Kim", "Myung-Sun Kim", "Gabbine Wee", "Choong-il Lee", "Hyongbum Kim", "Jin-Soo Kim"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0056476.t001", "stats"=>{"downloads"=>6, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Efficiencies_of_mutant_cell_enrichment_via_different_reporter_systems_/665267", "title"=>"Efficiencies of mutant cell enrichment via different reporter systems.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-02-18 01:27:47"}
  • {"files"=>["https://ndownloader.figshare.com/files/486668", "https://ndownloader.figshare.com/files/486674", "https://ndownloader.figshare.com/files/486677", "https://ndownloader.figshare.com/files/486680", "https://ndownloader.figshare.com/files/486683", "https://ndownloader.figshare.com/files/486685"], "description"=>"<div><p>The ability to enrich cells with targeted mutations greatly facilitates the process of using engineered nucleases, including zinc-finger nucleases and transcription activator-like effector nucleases, to construct such cells. We previously used surrogate reporters to enrich cells containing nuclease-induced mutations via flow cytometry. This method is, however, limited by the availability of flow cytometers. Furthermore, sorted cells occasionally fail to form colonies after exposure to a strong laser and hydrostatic pressure. Here we describe two different types of novel reporters that enable mutant cell enrichment without the use of flow cytometers. We designed reporters that express H-2K<sup>k</sup>, a surface antigen, and the hygromycin resistance protein (Hygro<sup>R</sup>), respectively, when insertions or deletions are generated at the target sequences by the activity of engineered nucleases. After cotransfection of these reporters and the engineered nuclease-encoding plasmids, H-2K<sup>k</sup>- and Hygro<sup>R</sup>-expressing cells were isolated using magnetic separation and hygromycin treatment, respectively. We found that mutant cells were drastically enriched in the isolated cells, suggesting that these two reporters enable efficient enrichment of mutants. We propose that these two reporters will greatly facilitate the use of engineered nucleases in a wider range of biomedical research.</p> </div>", "links"=>[], "tags"=>["antibiotics", "enrichment", "cells", "mutations"], "article_id"=>157302, "categories"=>["Biotechnology", "Biochemistry", "Genetics", "Biological Sciences"], "users"=>["Hyojin Kim", "Myung-Sun Kim", "Gabbine Wee", "Choong-il Lee", "Hyongbum Kim", "Jin-Soo Kim"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0056476.s001", "https://dx.doi.org/10.1371/journal.pone.0056476.s002", "https://dx.doi.org/10.1371/journal.pone.0056476.s003", "https://dx.doi.org/10.1371/journal.pone.0056476.s004", "https://dx.doi.org/10.1371/journal.pone.0056476.s005", "https://dx.doi.org/10.1371/journal.pone.0056476.s006"], "stats"=>{"downloads"=>19, "page_views"=>20, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Magnetic_Separation_and_Antibiotics_Selection_Enable_Enrichment_of_Cells_with_ZFN_TALEN_Induced_Mutations__/157302", "title"=>"Magnetic Separation and Antibiotics Selection Enable Enrichment of Cells with ZFN/TALEN-Induced Mutations", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-02-18 02:01:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/486985"], "description"=>"<p>(A) Enrichment of GFP<sup>+</sup> cells after magnetic separation. Scale bar = 50 µm. (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 indicates that each clone was detected once. Mutation frequencies were calculated by dividing the number of mutant clones by the number of total clones.</p>", "links"=>[], "tags"=>["separation-mediated", "enrichment", "cells", "episomal"], "article_id"=>157497, "categories"=>["Biotechnology", "Biochemistry", "Genetics", "Biological Sciences"], "users"=>["Hyojin Kim", "Myung-Sun Kim", "Gabbine Wee", "Choong-il Lee", "Hyongbum Kim", "Jin-Soo Kim"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0056476.g002", "stats"=>{"downloads"=>2, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Magnetic_separation_mediated_enrichment_of_CCR5_disrupted_cells_using_an_episomal_reporter_/157497", "title"=>"Magnetic separation-mediated enrichment of <i>CCR5</i>-disrupted cells using an episomal reporter.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-18 02:04:57"}
  • {"files"=>["https://ndownloader.figshare.com/files/487425"], "description"=>"<p>After <i>CCR5</i>-targeting ZFN (Z891) treatment, the mutant cells were selected by hygromycin treatment. The selected cells were plated at a density of 3,000 cells/100 mm dish, and the clonal colonies were manually picked 10 days after plating. The genomic DNA was isolated from the clonal colonies and analyzed. DNA sequences of the wild-type (WT) and mutant clones are shown, with ZFN recognition sites underlined, deleted bases indicated by dashes, and inserted bases in lower case. The number of occurrences is shown in parentheses; X1 and X2 indicate the number of each clone. Mutation frequencies were calculated by dividing the number of mutant clones by the number of total clones.</p>", "links"=>[], "tags"=>["hygromycin-selected"], "article_id"=>157947, "categories"=>["Biotechnology", "Biochemistry", "Genetics", "Biological Sciences"], "users"=>["Hyojin Kim", "Myung-Sun Kim", "Gabbine Wee", "Choong-il Lee", "Hyongbum Kim", "Jin-Soo Kim"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0056476.g006", "stats"=>{"downloads"=>1, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Clonal_analysis_of_hygromycin_selected_colonies_/157947", "title"=>"Clonal analysis of hygromycin-selected colonies.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-18 02:12:27"}
  • {"files"=>["https://ndownloader.figshare.com/files/487095"], "description"=>"<p>Nuclease-driven mutations were 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 gels indicate mutation percentages calculated by band intensities.</p>", "links"=>[], "tags"=>["reporter-mediated", "separations", "enrich", "zfn-", "talen-driven", "mutant"], "article_id"=>157598, "categories"=>["Biotechnology", "Biochemistry", "Genetics", "Biological Sciences"], "users"=>["Hyojin Kim", "Myung-Sun Kim", "Gabbine Wee", "Choong-il Lee", "Hyongbum Kim", "Jin-Soo Kim"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0056476.g003", "stats"=>{"downloads"=>2, "page_views"=>20, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Surrogate_reporter_mediated_magnetic_separations_enrich_ZFN_and_TALEN_driven_mutant_cells_/157598", "title"=>"Surrogate reporter-mediated magnetic separations enrich ZFN- and TALEN-driven mutant cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-18 02:06:38"}

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Relative Metric

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