An improved Red/ET recombineering system and mouse ES cells culture conditions for the generation of targeted mutant mice

• KUMAGAI Katsuyoshi

  Pre-Clinical Research Center, University-related Facilities, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan

• TAKANASHI Masakatsu

  Department of Pathology, Tokyo Medical University, Tokyo, Japan

• OHNO Shin-ichiro

  Department of Pathology, Tokyo Medical University, Tokyo, Japan

• KURODA Masahiko

  Department of Pathology, Tokyo Medical University, Tokyo, Japan

• SUDO Katsuko

  Pre-Clinical Research Center, University-related Facilities, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan

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.

Journal

• Experimental Animals

  Experimental Animals 66 (2), 125-136, 2017

  Japanese Association for Laboratory Animal Science

Keywords

• C57BL/6
• ES cells
• gene retrieval
• gene targeting
• KSR

Details

• CRID: 1390282680021251328
• NII Article ID: 130005635561
• NII Book ID: AA11032321
• DOI: 10.1538/expanim.16-0075
• ISSN: 18817122
• ISSN: 00075124
• ISSN: 13411357
• NDL BIB ID: 028109930
• PubMed: 27890869
• Web Site: https://ndlsearch.ndl.go.jp/books/R000000004-I028109930
• Web Site: https://www.jstage.jst.go.jp/article/expanim/66/2/66_16-0075/_pdf
• Web Site: https://search.jamas.or.jp/link/ui/2017298625
• Text Lang: en
• Data Source:
  • JaLC
  • NDL
  • Crossref
  • PubMed
  • CiNii Articles
  • KAKEN
• Abstract License Flag: Disallowed