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Title: Targeted gene integration using the combination of a sequence-specific DNA-binding protein and phiC31 integrase.
Authors: Nakanishi, Hideyuki
Higuchi, Yuriko  kyouindb  KAKEN_id  orcid (unconfirmed)
Yamashita, Fumiyoshi  kyouindb  KAKEN_id  orcid (unconfirmed)
Hashida, Mitsuru  kyouindb  KAKEN_id
Author's alias: 中西, 秀之
樋口, ゆり子
山下, 富義
橋田, 充
Keywords: Genomic integration
Site-specific integration
DNA-binding protein
Gene therapy
Issue Date: 17-Jul-2014
Publisher: Elsevier BV
Journal title: Journal of biotechnology
Volume: 186
Start page: 139
End page: 147
Abstract: 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.
Rights: © 2014 Elsevier B.V.
This is not the published version. Please cite only the published version.
DOI(Published Version): 10.1016/j.jbiotec.2014.07.012
PubMed ID: 25038544
Appears in Collections:Journal Articles

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