Multiple expression cassette exchange via TP901-1, R4, and Bxb1 integrase systems on a mouse artificial chromosome

Kosuke Tomimatsu, Kenji Kokura, Tadashi Nishida, Yuki Yoshimura, Yasuhiro Kazuki, Masashi Narita, Mitsuo Oshimura, Tetsuya Ohbayashi

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)

Abstract

The site-specific excision of a target DNA sequence for genetic knockout or lineage tracing is a powerful tool for investigating biological systems. Currently, site-specific recombinases (SSRs), such as Cre or Flp recombination target cassettes, have been successfully excised or inverted by a single SSR to regulate transgene expression. However, the use of a single SSR might restrict the complex control of gene expression. This study investigated the potential for expanding the multiple regulation of transgenes using three different integrase systems (TP901-1, R4, and Bxb1). We designed three excision cassettes that expressed luciferase, where the luciferase expression could be exchanged to a fluorescent protein by site-specific recombination. Individual cassettes that could be regulated independently by a different integrase were connected in tandem and inserted into a mouse artificial chromosome (MAC) vector in Chinese hamster ovary cells. The transient expression of an integrase caused the targeted luciferase activity to be lost and fluorescence was activated. Additionally, the integrase system enabled the specific excision of targeted DNA sequences without cross-reaction with the other recombination targets. These results suggest that the combined use of these integrase systems in a defined locus on a MAC vector permits the multiple regulation of transgene expression and might contribute to genomic or cell engineering.

Original languageEnglish
Pages (from-to)306-317
Number of pages12
JournalFEBS Open Bio
Volume7
Issue number3
DOIs
Publication statusPublished - Mar 1 2017
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)

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