Kenner Oliver, Kneisel Andrea, Klingler Jürgen, Bartelt Britta, Speit Günter, Vogel Walther, Kaufmann Dieter
Department of Human Genetics, University of Ulm, Albert-Einstein-Allee 11, D 89070 Ulm, Germany.
Biochem Biophys Res Commun. 2002 Dec 20;299(5):787-92. doi: 10.1016/s0006-291x(02)02749-3.
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.
通过特定寡核苷酸对真核细胞基因中的单个碱基进行靶向校正，是一项仍具争议的技术。在此，我们引入次黄嘌呤 - 鸟嘌呤 - 磷酸核糖转移酶（HPRT）基因中的点突变校正，作为测试靶向基因校正的另一个模型系统。在人类中，Hprt突变会导致莱施 - 奈恩综合征。我们使用仓鼠V79细胞，生成了三个分别带有一个hprt点突变的细胞系。用特定的45个碱基的硫代磷酸修饰单链寡核苷酸处理这些细胞系，并通过HAT培养基进行筛选。对存活的克隆进行研究，以检测各自hprt突变的校正情况。用寡核苷酸处理成功修复了所有三个hprt突变（hprt cDNA位置74，C→T；位置151，C→T；以及位置400，G→A）。校正效率非常低，但具有可重复性。我们认为，该系统能够让人依据靶序列和所用寡核苷酸来研究靶向基因校正。
