Targeted gene integration using the combination of a sequence-specific DNA-binding protein and phiC31 integrase.
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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.
収録刊行物
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- Journal of biotechnology
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Journal of biotechnology 186 139-147, 2014-07-17
Elsevier BV
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詳細情報 詳細情報について
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- CRID
- 1050564285747834368
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- NII論文ID
- 120005466731
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- NII書誌ID
- AA10458361
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- ISSN
- 18734863
- 01681656
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- HANDLE
- 2433/189465
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- 本文言語コード
- en
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- 資料種別
- journal article
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- データソース種別
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- IRDB
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