Generierung und Korrektur von Punktmutationen im HPRT-Gen von Säugerzellen mittels modifizierter einzelsträngiger DNA-Oligonukleotide

Abstract

Targeted correction of a single base in a gene of an eucaryotic cell by specific oligonucleotides is a yet controversial technique. Here, the correction of point mutations is introduced 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, three cell lines were generated with one hprt point mutation each. These cell lines were treated with specific single-stranded DNA oligonucleotides of 25 and 45 base in length modified with phosphothioate backbones on the four outer bases on each side. The respective hprt point mutation is positioned in the middle of the 45 base single-stranded oligonucleotide which is homologous to the non-transcribed strand of the wildtype hprt. The cells were selected by HAT medium, and 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, with a range from 0,86 to 3,61 successful hprt corrected cells/10^6 cells. It is suggested that this system allows one to investigate targeted gene correction in dependence on the target sequence and the oligonucleotides used.