title: Rapid tagging of endogenous mouse genes by recombineering and ES cell complementation of tetraploid blastocysts.
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.
authors.affiliation: Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
authors.forename: Dewang
authors.initials: D
authors.lastname: Zhou
authors.forename: Jin-Xiang
authors.initials: JX
authors.lastname: Ren
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Ryan
authors.forename: N Patrick
authors.initials: NP
authors.lastname: Higgins
authors.forename: Tim M
authors.initials: TM
authors.lastname: Townes
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000648:ultrastructure; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial; D019976:Cloning, Organism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology*; D004926:Escherichia coli / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D005816:Genetic Complementation Test; D007267:Injections; D051741:Kruppel-Like Transcription Factors; D051379:Mice / Q000235:genetics*; D011123:Polyploidy; D011995:Recombination, Genetic; D013234:Stem Cells; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D051741:Kruppel-Like Transcription Factors; D014157:Transcription Factors; C080774:erythroid Kruppel-like factor