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Using this strategy, we previously knocked-in the EGFP coding sequence into the N-terminal region of the beta-actin gene specifically in the pyramidal neurons in layer 2/3 of the somatosensory cortex. However, the knock-in efficiency was less than 2% of the transfected neurons. In this study, we sought to improve the knock-in efficiency using this system. First, we varied the length of the homology arms of the beta-actin donor template DNA, and found that the knock-in efficiency was increased to similar to 14% by extending the length of the 5\u0027 and 3\u0027 homology arms to 1.6 kb and 2.0 kb, respectively. We then tested the effect of the DNA repair protein RAD51 and the knock-in efficiency was increased up to 2.5-fold when co-transfecting with two different beta-actin and a camk2a targeting EGFP knock-in modules. The RAD51 overexpression did not alter the migration of developing neurons, density or morphology of the dendritic spines compared to those in neurons not transfected with RAD51. RAD51 expression will be useful for increasing the knock-in efficiency in neurons in vivo by CRISPR/Cas9-mediated homology directed repair (HDR). (C) 2020 Elsevier Inc. 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DNA repair protein RAD51 enhances the CRISPR/Cas9-mediated knock-in efficiency in brain neurons (DNA修復タンパク質RAD51は脳の神経細胞でのCRISPR/Cas9を介した遺伝子ノックイン効率を上昇させる。)
http://hdl.handle.net/10091/00022173
http://hdl.handle.net/10091/00022173c50c9dc7-3bd0-4818-8047-fe8207c7367c
名前 / ファイル | ライセンス | アクション |
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Item type | 学位論文 / Thesis or Dissertation(1) | |||||
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公開日 | 2020-06-15 | |||||
タイトル | ||||||
言語 | en | |||||
タイトル | DNA repair protein RAD51 enhances the CRISPR/Cas9-mediated knock-in efficiency in brain neurons (DNA修復タンパク質RAD51は脳の神経細胞でのCRISPR/Cas9を介した遺伝子ノックイン効率を上昇させる。) | |||||
言語 | ||||||
言語 | eng | |||||
資源タイプ | ||||||
資源 | http://purl.org/coar/resource_type/c_db06 | |||||
タイプ | doctoral thesis | |||||
アクセス権 | ||||||
アクセス権 | open access | |||||
アクセス権URI | http://purl.org/coar/access_right/c_abf2 | |||||
著者 |
栗原, 大河
× 栗原, 大河 |
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出版者 | ||||||
出版者 | 信州大学 | |||||
引用 | ||||||
内容記述タイプ | Other | |||||
内容記述 | 栗原 大河. DNA repair protein RAD51 enhances the CRISPR/Cas9-mediated knock-in efficiency in brain neurons (DNA修復タンパク質RAD51は脳の神経細胞でのCRISPR/Cas9を介した遺伝子ノックイン効率を上昇させる。). 信州大学, 2020, 博士論文. | |||||
書誌情報 |
発行日 2020-03-31 |
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学位授与番号 | ||||||
学位授与番号 | 13601甲第1206号 | |||||
学位授与年月日 | ||||||
学位授与年月日 | 2020-03-31 | |||||
学位名 | ||||||
学位名 | 博士(医学) | |||||
学位授与機関 | ||||||
学位授与機関識別子Scheme | kakenhi | |||||
学位授与機関識別子 | 13601 | |||||
学位授与機関名 | 信州大学(Shinshu university) | |||||
学位の区分 | ||||||
doctoral | ||||||
学位の分野 | ||||||
医学 | ||||||
学位の報告番号 | ||||||
甲第1206号 | ||||||
内容記述 | ||||||
内容記述タイプ | Other | |||||
内容記述 | 雑誌に発表。BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS. 524(3):621-628 (2020); doi:10.1016/j.bbrc.2020.01.132. | |||||
資源タイプ(コンテンツの種類) | ||||||
内容記述タイプ | Other | |||||
内容記述 | Thesis | |||||
PubMed | ||||||
識別子タイプ | PMID | |||||
関連識別子 | https://pubmed.ncbi.nlm.nih.gov/32029273/ | |||||
関連名称 | 32029273 | |||||
DOI | ||||||
識別子タイプ | DOI | |||||
関連識別子 | https://doi.org/10.1016/j.bbrc.2020.01.132 | |||||
関連名称 | 10.1016/j.bbrc.2020.01.132 | |||||
権利 | ||||||
権利情報 | © 2020. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ | |||||
出版タイプ | ||||||
出版タイプ | NA | |||||
出版タイプResource | http://purl.org/coar/version/c_be7fb7dd8ff6fe43 | |||||
WoS | ||||||
Web of Science | ||||||
URL | http://gateway.isiknowledge.com/gateway/Gateway.cgi?&GWVersion=2&SrcAuth=ShinshuUniv&SrcApp=ShinshuUniv&DestLinkType=FullRecord&DestApp=WOS&KeyUT=000526792800014 |