title: Exploring new gene integration sites for gene knock-in by gene-trapping strategy.
abstract: The knock-in mouse is a powerful tool for biological research, but the stability of expression of an integrated gene strongly depends on where it is integrated in the mouse genome. At present, there are an insufficient number of loci suitable for gene knock-in, such as the Rosa26 locus. Therefore, in this study, we developed an efficient strategy for identifying genome loci suitable for gene knock-in and characterized the properties of such loci for gene integration. For efficient discovery and characterization, we constructed a new gene-trapping vector that enables monitoring of the expression of both trapped and integrated genes using fluorescence. We successfully obtained fluorescent-positive mouse embryonic stem cell (mESC) clones with the vector. Thorough analysis of the expression of fluorescent proteins in chimera embryos generated with the obtained mESC clones, some of the gene-trapped chimera embryos showed stable and ubiquitous expression of the integrated gene. Furthermore, adult mice derived from one of the gene-trapped mESC clones showed ubiquitous expression of the integrated gene in various tissues without any unusual phenotype. This indicated that the identified locus possesses high potential for foreign gene integration. Our strategy allows for efficient discovery and characterization of mouse genome loci for gene integration.
authors.affiliation: Division of Molecular Genetics and Biofunction, Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Yonago, Japan.
authors.forename: Isamu
authors.initials: I
authors.lastname: Nanchi
authors.forename: Yuki
authors.initials: Y
authors.lastname: Yoshimura
authors.forename: Kazuomi
authors.initials: K
authors.lastname: Nakamura
authors.forename: Yusaku
authors.initials: Y
authors.lastname: Masago
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Ohbayashi
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Okuda
mesh_terms: D000818:Animals; D001483:Base Sequence; D001755:Blastocyst / Q000502:physiology; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology / Q000502:physiology*; D005260:Female; D005456:Fluorescent Dyes; D055879:Gene Knock-In Techniques / Q000379:methods*; D056426:Genetic Loci; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008969:Molecular Sequence Data
chemical_list: D005456:Fluorescent Dyes; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
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