Article

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

Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Nucleic Acids Research (impact factor: 8.03). 02/2004; 32(16):e128. DOI:10.1093/nar/gnh128 pp.e128
Source: PubMed

ABSTRACT 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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Keywords

bacterial artificial chromosome clones
 
endogenous genes
 
ES cells
 
gap-repair
 
injecting genetically
 
Knockin vectors
 
large-scale TAP tagging
 
mammalian genes
 
modified cells
 
recombineering
 
tandem affinity purification
 
TAP tag
 
TAP)/floxed marker gene plasmid
 
tetraploid blastocyst complementation
 
tetraploid blastocysts
 
timeline