title: An improved Red/ET recombineering system and mouse ES cells culture conditions for the generation of targeted mutant mice.
abstract: Targeted mutant mice generated on a C57BL/6 background are powerful tools for analysis of the biological functions of genes, and gene targeting technologies using mouse embryonic stem (ES) cells have been used to generate such mice. Recently, a bacterial artificial chromosome (BAC) recombineering system was established for the construction of targeting vectors. However, gene retrieval from BACs for the generation of gene targeting vectors using this system remains difficult. Even when construction of a gene targeting vector is successful, the efficiency of production of targeted mutant mice from ES cells derived from C57BL/6 mice are poor. Therefore, in this study, we first improved the strategy for the retrieval of genes from BACs and their transfer into a DT-A plasmid, for the generation of gene targeting vectors using the BAC recombineering system. Then, we attempted to generate targeted mutant mice from ES cell lines derived from C57BL/6 mice, by culturing in serum-free medium. In conclusion, we established an improved strategy for the efficient generation of targeted mutant mice on a C57BL/6 background, which are useful for the in vivo analysis of gene functions and regulation.
authors.affiliation: Pre-Clinical Research Center, University-related Facilities, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan.
authors.forename: Katsuyoshi
authors.initials: K
authors.lastname: Kumagai
authors.affiliation: Department of Pathology, Tokyo Medical University, Tokyo, Japan.
authors.forename: Masakatsu
authors.initials: M
authors.lastname: Takanashi
authors.affiliation: Department of Pathology, Tokyo Medical University, Tokyo, Japan.
authors.forename: Shin-Ichiro
authors.initials: SI
authors.lastname: Ohno
authors.affiliation: Department of Pathology, Tokyo Medical University, Tokyo, Japan.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Kuroda
authors.affiliation: Pre-Clinical Research Center, University-related Facilities, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan.
authors.forename: Katsuko
authors.initials: K
authors.lastname: Sudo
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D016895:Culture Media, Serum-Free; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D000066450:Mouse Embryonic Stem Cells; D010957:Plasmids; D011995:Recombination, Genetic
chemical_list: D016895:Culture Media, Serum-Free
keywords: C57BL/6; ES cells; KSR; gene retrieval; gene targeting