title: Construction and applications of exon-trapping gene-targeting vectors with a novel strategy for negative selection.
abstract: BACKGROUND
  Targeted gene modification by homologous recombination provides a powerful tool for studying gene function in cells and animals. In higher eukaryotes, non-homologous integration of targeting vectors occurs several orders of magnitude more frequently than does targeted integration, making the gene-targeting technology highly inefficient. For this reason, negative-selection strategies have been employed to reduce the number of drug-resistant clones associated with non-homologous vector integration, particularly when artificial nucleases to introduce a DNA break at the target site are unavailable or undesirable. As such, an exon-trap strategy using a promoterless drug-resistance marker gene provides an effective way to counterselect non-homologous integrants. However, constructing exon-trapping targeting vectors has been a time-consuming and complicated process.
  
  
  RESULTS
  By virtue of highly efficient att-mediated recombination, we successfully developed a simple and rapid method to construct plasmid-based vectors that allow for exon-trapping gene targeting. These exon-trap vectors were useful in obtaining correctly targeted clones in mouse embryonic stem cells and human HT1080 cells. Most importantly, with the use of a conditionally cytotoxic gene, we further developed a novel strategy for negative selection, thereby enhancing the efficiency of counterselection for non-homologous integration of exon-trap vectors.
  
  
  CONCLUSIONS
  Our methods will greatly facilitate exon-trapping gene-targeting technologies in mammalian cells, particularly when combined with the novel negative selection strategy.
authors.affiliation: Graduate School of Nanobioscience, Yokohama City University, Yokohama, 236-0027, Japan. n115352g@yokohama-cu.ac.jp.
authors.forename: Shinta
authors.initials: S
authors.lastname: Saito
authors.affiliation: Graduate School of Science, Chiba University, Chiba, 263-8522, Japan. kiyoeura@chiba-u.jp.
authors.forename: Kiyoe
authors.initials: K
authors.lastname: Ura
authors.affiliation: Graduate School of Nanobioscience, Yokohama City University, Yokohama, 236-0027, Japan. i080266e@gmail.com.
authors.forename: Miho
authors.initials: M
authors.lastname: Kodama
authors.affiliation: Graduate School of Nanobioscience, Yokohama City University, Yokohama, 236-0027, Japan. nadachi@yokohama-cu.ac.jp.|Advanced Medical Research Center, Yokohama City University, Yokohama, 236-0004, Japan. nadachi@yokohama-cu.ac.jp.
authors.forename: Noritaka
authors.initials: N
authors.lastname: Adachi
mesh_terms: D000818:Animals; D002460:Cell Line; D005091:Exons / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D051379:Mice; D013234:Stem Cells