2014年9月
Targeted gene integration using the combination of a sequence-specific DNA-binding protein and phiC31 integrase
JOURNAL OF BIOTECHNOLOGY
- ,
- ,
- ,
- 巻
- 186
- 号
- 開始ページ
- 139
- 終了ページ
- 147
- 記述言語
- 英語
- 掲載種別
- 研究論文(学術雑誌)
- DOI
- 10.1016/j.jbiotec.2014.07.012
- 出版者・発行元
- ELSEVIER SCIENCE BV
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. (C) 2014 Elsevier B.V. All rights reserved.
- リンク情報
- ID情報
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- DOI : 10.1016/j.jbiotec.2014.07.012
- ISSN : 0168-1656
- eISSN : 1873-4863
- PubMed ID : 25038544
- Web of Science ID : WOS:000343836000017