title: Multiple expression cassette exchange via TP901-1, R4, and Bxb1 integrase systems on a mouse artificial chromosome.
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.
authors.affiliation: Research Center for Bioscience and Technology Tottori University Yonago Japan; Japan Society for the Promotion of Science Tokyo Japan.
authors.forename: Kosuke
authors.initials: K
authors.lastname: Tomimatsu
authors.affiliation: Chromosome Engineering Research Center Tottori University Yonago Japan; Division of Human Genome Science Department of Molecular and Cellular Biology School of Life Sciences Faculty of Medicine Tottori University Yonago Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Kokura
authors.affiliation: Research Center for Bioscience and Technology Tottori University Yonago Japan.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Nishida
authors.affiliation: Department of Biomedical Science Institute of Regenerative Medicine and Biofunction Graduate School of Medical Sciences Tottori University Yonago Japan; Central Institute for Experimental Animals Kawasaki Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Yoshimura
authors.affiliation: Chromosome Engineering Research Center Tottori University Yonago Japan; Department of Biomedical Science Institute of Regenerative Medicine and Biofunction Graduate School of Medical Sciences Tottori University Yonago Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kazuki
authors.affiliation: Cancer Research UK Cambridge Institute Li Ka Shing Centre University of Cambridge UK.
authors.forename: Masashi
authors.initials: M
authors.lastname: Narita
authors.affiliation: Chromosome Engineering Research Center Tottori University Yonago Japan; Department of Biomedical Science Institute of Regenerative Medicine and Biofunction Graduate School of Medical Sciences Tottori University Yonago Japan.
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Oshimura
authors.affiliation: Research Center for Bioscience and Technology Tottori University Yonago Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Ohbayashi
keywords: fluorescent protein; integrase; lineage‐tracing reporter; luciferase; mouse artificial chromosome