Accumulative gene integration into a pre-determined site using Cre/loxP

Hirokazu Obayashi, Kawabe Yoshinori, Hirokatsu Makitsubo, Ryoko Watanabe, Yujiro Kameyama, Shuohao Huang, Yuta Takenouchi, Akira Ito, Masamichi Kamihira

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Site-specific gene recombination systems, such as Cre/. loxP, have been used for genetic modification of cells and organisms in both basic and applied research. We previously developed an accumulative gene integration system (AGIS), in which target gene cassettes could be repeatedly integrated into a pre-determined site on a plasmid or cellular genome by recombinase-mediated cassette exchange (RMCE), using Cre and mutated loxPs. In the present study, we designed a simplified AGIS. For gene integration into a target site, the previous system used two loxP sites in the acceptor DNA, whereas the new system uses a single loxP site. The gene integration reactions were repeated four times in vitro using Cre protein and specific plasmids. The expected integration reactions mediated by Cre occurred at the loxP sites, resulting in integration of four target genes. The system was also used for genomic integration of reporter genes using Chinese hamster ovary (CHO) cells. The reporter genes were efficiently introduced into the CHO genome in a Cre-dependent manner, and transgene expression was detected after the integration reaction. The expression levels of the reporter genes were enhanced, corresponding to the increase of transgene copy number. Recombinase-mediated AGIS provides a useful tool for the modification of cellular genomes.

Original languageEnglish
Pages (from-to)381-388
Number of pages8
JournalJournal of Bioscience and Bioengineering
Volume113
Issue number3
DOIs
Publication statusPublished - Mar 1 2012

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Genes
Systems Integration
Reporter Genes
Recombinases
Genome
Cricetulus
Transgenes
Ovary
Plasmids
Genetic Recombination
DNA
Research
Cells
Proteins

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Cite this

Accumulative gene integration into a pre-determined site using Cre/loxP. / Obayashi, Hirokazu; Yoshinori, Kawabe; Makitsubo, Hirokatsu; Watanabe, Ryoko; Kameyama, Yujiro; Huang, Shuohao; Takenouchi, Yuta; Ito, Akira; Kamihira, Masamichi.

In: Journal of Bioscience and Bioengineering, Vol. 113, No. 3, 01.03.2012, p. 381-388.

Research output: Contribution to journalArticle

Obayashi, Hirokazu ; Yoshinori, Kawabe ; Makitsubo, Hirokatsu ; Watanabe, Ryoko ; Kameyama, Yujiro ; Huang, Shuohao ; Takenouchi, Yuta ; Ito, Akira ; Kamihira, Masamichi. / Accumulative gene integration into a pre-determined site using Cre/loxP. In: Journal of Bioscience and Bioengineering. 2012 ; Vol. 113, No. 3. pp. 381-388.
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