|Title:||Targeted gene integration using the combination of a sequence-specific DNA-binding protein and phiC31 integrase.|
Higuchi, Yuriko https://orcid.org/0000-0002-9869-1019 (unconfirmed)
Yamashita, Fumiyoshi https://orcid.org/0000-0002-3503-8696 (unconfirmed)
|Author's alias:||中西, 秀之|
|Journal title:||Journal of biotechnology|
|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.
|Appears in Collections:||Journal Articles|
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