title: Targeted gene correction of hprt mutations by 45 base single-stranded oligonucleotides.
abstract: Targeted correction of a single base in a gene of an eucaryotic cell by specific oligonucleotides is a yet controversial technique. Here, we introduce the correction of point mutations in the hypoxanthine-guanine-phosphoribosyl-transferase (HPRT) gene as an additional model system to test targeted gene correction. In human, Hprt mutations cause Lesch-Nyhan syndrome. Using hamster V79 cells, we generated three cell lines with one hprt point mutation each. These cell lines were treated with specific single-stranded 45 base phosphothioate modified oligonucleotides and selected by HAT medium. The surviving clones were investigated for the correction of the respective hprt mutation. Treatment with the oligonucleotides was successful in repairing all three hprt mutations (hprt cDNA position 74, C --> T; position 151, C --> T; and position 400, G --> A). The correction efficiency was very low but reproducible. We suggest that this system allows one to investigate targeted gene correction in dependence on the target sequence and the oligonucleotides used.
authors.affiliation: Department of Human Genetics, University of Ulm, Albert-Einstein-Allee 11, D 89070 Ulm, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Kenner
authors.forename: Andrea
authors.initials: A
authors.lastname: Kneisel
authors.forename: Jürgen
authors.initials: J
authors.lastname: Klingler
authors.forename: Britta
authors.initials: B
authors.lastname: Bartelt
authors.forename: Günter
authors.initials: G
authors.lastname: Speit
authors.forename: Walther
authors.initials: W
authors.lastname: Vogel
authors.forename: Dieter
authors.initials: D
authors.lastname: Kaufmann
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002999:Clone Cells; D006224:Cricetinae; D004277:DNA, Single-Stranded; D018390:Gene Targeting / Q000379:methods*; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D009841:Oligonucleotides / Q000737:chemistry; D017354:Point Mutation
chemical_list: D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D007041:Hypoxanthine Phosphoribosyltransferase