{"_buckets": {"deposit": "998d8770-8584-43b7-996f-6163450ed474"}, "_deposit": {"id": "20845", "owners": [], "pid": {"revision_id": 0, "type": "depid", "value": "20845"}, "status": "published"}, "_oai": {"id": "oai:soar-ir.repo.nii.ac.jp:00020845", "sets": ["462"]}, "author_link": ["108319", "108320", "108321", "108322", "108323", "108324", "108325", "108326", "108327", "108328"], "item_1628147817048": {"attribute_name": "出版タイプ", "attribute_value_mlt": [{"subitem_version_resource": "http://purl.org/coar/version/c_ab4af688f83e57aa", "subitem_version_type": "AM"}]}, "item_6_biblio_info_6": {"attribute_name": "書誌情報", "attribute_value_mlt": [{"bibliographicIssueDates": {"bibliographicIssueDate": "2018-03-01", "bibliographicIssueDateType": "Issued"}, "bibliographicPageEnd": "38", "bibliographicPageStart": "29", "bibliographicVolumeNumber": "108", "bibliographic_titles": [{"bibliographic_title": "THERIOGENOLOGY"}]}]}, "item_6_description_20": {"attribute_name": "抄録", "attribute_value_mlt": [{"subitem_description": "Recently, successful one-step genome editing by microinjection of CRISPR/Cas9-related mRNA components into the porcine zygote has been described. Given the relatively long gestational period and the high cost of housing swine, the establishment of an effective microinjection-based porcine genome editing method is urgently required. Previously, we have attempted to disrupt a gene encoding alpha-1,3-galactosyltransferase (GGTA1), which synthesizes the alpha-Gal epitope, by microinjecting CRISPR/Cas9-related nucleic acids and enhanced green fluorescent protein (EGFP) mRNA into porcine oocytes immediately after electrical activation. We found that genome editing was indeed induced, although the resulting blastocysts were mosaic and the frequency of modified cells appeared to be low (50%). To improve genome editing efficiency in porcine oocytes, cytoplasmic injection was performed 6 h after electrical activation, a stage wherein the pronucleus is formed. The developing blastocysts exhibited higher levels of EGFP. Furthermore, the T7 endonuclease 1 assay and subsequent sequencing demonstrated that these embryos exhibited increased genome editing efficiencies (69%), although a high degree of mosaicism for the induced mutation was still observed. Single blastocyst-based cytochemical staining with fluorescently labeled isolectin BS-I-B-4 also confirmed this mosaicism. Thus, the development of a technique that avoids or reduces such mosaicism would be a key factor for efficient knock out piglet production via microinjection. (C) 2017 Elsevier Inc. 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This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/"}]}, "item_6_select_64": {"attribute_name": "著者版フラグ", "attribute_value_mlt": [{"subitem_select_item": "author"}]}, "item_6_source_id_35": {"attribute_name": "ISSN", "attribute_value_mlt": [{"subitem_source_identifier": "0093-691X", "subitem_source_identifier_type": "PISSN"}]}, "item_6_source_id_39": {"attribute_name": "NII ISSN", "attribute_value_mlt": [{"subitem_source_identifier": "0093-691X", "subitem_source_identifier_type": "PISSN"}]}, "item_6_source_id_40": {"attribute_name": "書誌レコードID", "attribute_value_mlt": [{"subitem_source_identifier": "AA00862972", "subitem_source_identifier_type": "NCID"}]}, "item_6_text_69": {"attribute_name": "wosonly authkey", "attribute_value_mlt": [{"subitem_text_value": "ONE-STEP GENERATION; HIGHLY-ACTIVE SGRNAS; CRISPR-CAS SYSTEMS; ALPHA-GAL EPITOPE; IN-VITRO; GENE; EMBRYOS; CELLS; MUTATIONS; MICE"}]}, "item_6_text_70": {"attribute_name": "wosonly keywords", "attribute_value_mlt": [{"subitem_text_value": "Recently, successful one-step genome editing by microinjection of CRISPR/Cas9-related mRNA components into the porcine zygote has been described. Given the relatively long gestational period and the high cost of housing swine, the establishment of an effective microinjection-based porcine genome editing method is urgently required. Previously, we have attempted to disrupt a gene encoding alpha-1,3-galactosyltransferase (GGTA1), which synthesizes the alpha-Gal epitope, by microinjecting CRISPR/Cas9-related nucleic acids and enhanced green fluorescent protein (EGFP) mRNA into porcine oocytes immediately after electrical activation. We found that genome editing was indeed induced, although the resulting blastocysts were mosaic and the frequency of modified cells appeared to be low (50%). To improve genome editing efficiency in porcine oocytes, cytoplasmic injection was performed 6 h after electrical activation, a stage wherein the pronucleus is formed. The developing blastocysts exhibited higher levels of EGFP. Furthermore, the T7 endonuclease 1 assay and subsequent sequencing demonstrated that these embryos exhibited increased genome editing efficiencies (69%), although a high degree of mosaicism for the induced mutation was still observed. Single blastocyst-based cytochemical staining with fluorescently labeled isolectin BS-I-B-4 also confirmed this mosaicism. Thus, the development of a technique that avoids or reduces such mosaicism would be a key factor for efficient knock out piglet production via microinjection. (C) 2017 Elsevier Inc. All rights reserved."}]}, "item_6_textarea_68": {"attribute_name": "wosonly abstract", "attribute_value_mlt": [{"subitem_textarea_value": "alpha-Gal epitope; alpha-1,3-Galactosyltransferase; CRISPR/Cas9; Microinjection; mRNA; Mosaicism; Biallelic KO; Indels; Isolectin BS-I-B-4"}]}, "item_creator": {"attribute_name": "著者", "attribute_type": "creator", "attribute_value_mlt": [{"creatorNames": [{"creatorName": "Sato, Masahiro", "creatorNameLang": "en"}], "nameIdentifiers": [{"nameIdentifier": "108319", "nameIdentifierScheme": "WEKO"}]}, {"creatorNames": [{"creatorName": "Kosuke, Maeda", "creatorNameLang": "en"}], "nameIdentifiers": [{"nameIdentifier": "108320", "nameIdentifierScheme": "WEKO"}]}, {"creatorNames": [{"creatorName": "Koriyama, Miyu", "creatorNameLang": "en"}], "nameIdentifiers": [{"nameIdentifier": "108321", "nameIdentifierScheme": "WEKO"}]}, {"creatorNames": [{"creatorName": "Inada, Emi", "creatorNameLang": "en"}], "nameIdentifiers": [{"nameIdentifier": "108322", "nameIdentifierScheme": "WEKO"}]}, {"creatorNames": [{"creatorName": "Saitoh, Issei", "creatorNameLang": "en"}], "nameIdentifiers": [{"nameIdentifier": "108323", "nameIdentifierScheme": "WEKO"}]}, {"creatorNames": [{"creatorName": "Ohtsuka, Masato", "creatorNameLang": "en"}], "nameIdentifiers": [{"nameIdentifier": "108324", "nameIdentifierScheme": "WEKO"}]}, {"creatorNames": [{"creatorName": "Nakamura, Shingo", "creatorNameLang": "en"}], "nameIdentifiers": [{"nameIdentifier": "108325", "nameIdentifierScheme": "WEKO"}]}, {"creatorNames": [{"creatorName": "Sakurai, Takayuki", "creatorNameLang": "en"}], "nameIdentifiers": [{"nameIdentifier": "108326", "nameIdentifierScheme": "WEKO"}]}, {"creatorNames": [{"creatorName": "Watanabe, Satoshi", "creatorNameLang": "en"}], "nameIdentifiers": [{"nameIdentifier": "108327", "nameIdentifierScheme": "WEKO"}]}, {"creatorNames": [{"creatorName": "Miyoshi, Kazuchika", "creatorNameLang": "en"}], "nameIdentifiers": [{"nameIdentifier": "108328", "nameIdentifierScheme": "WEKO"}]}]}, "item_files": {"attribute_name": "ファイル情報", "attribute_type": "file", "attribute_value_mlt": [{"accessrole": "open_date", "date": [{"dateType": "Available", "dateValue": "2019-09-09"}], "displaytype": "detail", "download_preview_message": "", "file_order": 0, "filename": "16K15233_01.pdf", "filesize": [{"value": "3.1 MB"}], "format": "application/pdf", "future_date_message": "", "is_thumbnail": false, "licensefree": "© 2018. 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