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Nucleic Acids Research 2004 32(16):e128; doi:10.1093/nar/gnh128
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Published online 8 September 2004

Nucleic Acids Research, Vol. 32 No. 16 © Oxford University Press 2004; all rights reserved

Rapid tagging of endogenous mouse genes by recombineering and ES cell complementation of tetraploid blastocysts

Dewang Zhou, Jin-Xiang Ren, Thomas M. Ryan, N. Patrick Higgins and Tim M. Townes*

Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, AL 35294, USA

* To whom correspondence should be addressed. Tel: +1 205 934 5294; Fax: +1 205 934 2889; Email: ttownes{at}uab.edu

Received July 16, 2004; Revised and Accepted August 20, 2004

The construction of knockin vectors designed to modify endogenous genes in embryonic stem (ES) cells and the generation of mice from these modified cells is time consuming. The timeline of an experiment from the conception of an idea to the availability of mature mice is at least 9 months. We describe a method in which this timeline is typically reduced to 3 months. Knockin vectors are rapidly constructed from bacterial artificial chromosome clones by recombineering followed by gap-repair (GR) rescue, and mice are rapidly derived by injecting genetically modified ES cells into tetraploid blastocysts. We also describe a tandem affinity purification (TAP)/floxed marker gene plasmid and a GR rescue plasmid that can be used to TAP tag any murine gene. The combination of recombineering and tetraploid blastocyst complementation provides a means for large-scale TAP tagging of mammalian genes.


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