論文

査読有り
2014年9月

Targeted gene integration using the combination of a sequence-specific DNA-binding protein and phiC31 integrase

JOURNAL OF BIOTECHNOLOGY
  • Hideyuki Nakanishi
  • ,
  • Yuriko Higuchi
  • ,
  • Fumiyoshi Yamashita
  • ,
  • Mitsuru Hashida

186
30
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.

Web of Science ® 被引用回数 : 2

リンク情報
DOI
https://doi.org/10.1016/j.jbiotec.2014.07.012
PubMed
https://www.ncbi.nlm.nih.gov/pubmed/25038544
Web of Science
https://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=JSTA_CEL&SrcApp=J_Gate_JST&DestLinkType=FullRecord&KeyUT=WOS:000343836000017&DestApp=WOS_CPL

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