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j.jbiotec.2014.07.012.pdf | 1.23 MB | Adobe PDF | 見る/開く |
タイトル: | Targeted gene integration using the combination of a sequence-specific DNA-binding protein and phiC31 integrase. |
著者: | Nakanishi, Hideyuki Higuchi, Yuriko https://orcid.org/0000-0002-9869-1019 (unconfirmed) Yamashita, Fumiyoshi https://orcid.org/0000-0002-3503-8696 (unconfirmed) Hashida, Mitsuru |
著者名の別形: | 中西, 秀之 樋口, ゆり子 山下, 富義 橋田, 充 |
キーワード: | Genomic integration Site-specific integration Integrase DNA-binding protein Gene therapy |
発行日: | 17-Jul-2014 |
出版者: | Elsevier BV |
誌名: | Journal of biotechnology |
巻: | 186 |
開始ページ: | 139 |
終了ページ: | 147 |
抄録: | PhiC31 integrase-based vectors can integrate therapeutic genes selectively into attP or pseudo-attP sites in genomes, but considerable numbers of pseudo-attP sites in human genomes exist inside endogenous gene-coding regions. To avoid endogenous gene disruptions, we aimed to enhance the integration site-specificity of the phiC31 integrase-based vector using a sequence-specific DNA-binding protein containing Gal4 and LexA DNA-binding motifs. The dual DNA-binding protein was designed to tether the UAS-containing donor vector to the target sequence, the LexA operator, and restrict integration to sites close to the LexA operator. To analyze the site-specificity in chromosomal integration, a human cell line having LexA operators on the genome was established, and the cell line was transfected with donor vectors expressing the DNA-binding protein and the phiC31 integrase expression vector (helper vector). Quantitative PCR indicated that integration around the LexA operator was 26-fold higher with the UAS-containing donor vector than with the control. Sequence analysis confirmed that the integration occurred around the LexA operator. The dual DNA-binding protein-based targeted integration strategy developed herein would allow safer and more reliable genetic manipulations for various applications, including gene and cell therapies. |
著作権等: | © 2014 Elsevier B.V. This is not the published version. Please cite only the published version. この論文は出版社版でありません。引用の際には出版社版をご確認ご利用ください。 |
URI: | http://hdl.handle.net/2433/189465 |
DOI(出版社版): | 10.1016/j.jbiotec.2014.07.012 |
PubMed ID: | 25038544 |
出現コレクション: | 学術雑誌掲載論文等 |
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