Targeted gene correction of hprt mutations by 45 base single-stranded oligonucleotides.
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.
Kenner
Oliver
O
Department of Human Genetics, University of Ulm, Albert-Einstein-Allee 11, D 89070 Ulm, Germany.
Kneisel
Andrea
A
Klingler
Jürgen
J
Bartelt
Britta
B
Speit
Günter
G
Vogel
Walther
W
Kaufmann
Dieter
D
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
D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D007041:Hypoxanthine Phosphoribosyltransferase