title: Targeted gene integration using the combination of a sequence-specific DNA-binding protein and phiC31 integrase.
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.
authors.affiliation: Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan; Japan Society for the Promotion of Science (JSPS), Sumitomo-Ichibancho FS Bldg., 8 Ichibancho, Chiyoda-ku, Tokyo 102-8472, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Nakanishi
authors.affiliation: Institute for Innovative NanoBio Drug Discovery and Development, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan; PRESTO, Japan Science and Technology Agency (JST), Kawaguchi Center Building 4-1-8, Honcho, Kawaguchi-shi, Saitama 332-0012, Japan.
authors.forename: Yuriko
authors.initials: Y
authors.lastname: Higuchi
authors.affiliation: Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Fumiyoshi
authors.initials: F
authors.lastname: Yamashita
authors.affiliation: Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan; Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, 69 Yoshida Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501, Japan. Electronic address: hashidam@pharm.kyoto-u.ac.jp.
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Hashida
mesh_terms: D001435:Bacteriophages / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D019426:Integrases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D017422:Sequence Analysis, DNA; D014162:Transfection / Q000379:methods*; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D014764:Viral Proteins; D019426:Integrases
keywords: DNA-binding protein; Gene therapy; Genomic integration; Integrase; Site-specific integration