Hellgren D, Luthman H, Lambert B
Department of Clinical Genetics, Karolinska Hospital, Stockholm, Sweden.
Mutat Res. 1989 Jan;210(1):197-206. doi: 10.1016/0027-5107(89)90059-6.
Homologous recombination between 2 truncated neo genes stably integrated in the genome of Chinese hamster ovary (CHO) cells was studied. A vector containing a functional gpt gene and 2 tandemly arranged G418 resistance (neo) gene fragments with about 400 bp of sequence homology was transfected into CHO cells. Clonal cell lines were established from transfected cultures and the spontaneous frequency of G418-resistant revertants was found to range between 1 x 10(-4) and 5 x 10(-4). The ability of the alkylating agents MMS and HN2 to induce recombination of the transfected neo genes was studied in 2 of the cell lines. After treatment with MMS at doses that reduced survival to 10% of the control these cell lines showed a dose-dependent increase in the frequency of G418-resistant revertants. No effect was observed after treatment with HN2. All G418-resistant subclones contained a new restriction fragment indicating that a whole neo gene had been formed by rearrangement in pairs of truncated neo genes. Hence, this system can be used to study molecular mechanisms and chemical inducibility of homologous recombination in mammalian cells.
对稳定整合在中国仓鼠卵巢（CHO）细胞基因组中的两个截短的新霉素基因之间的同源重组进行了研究。将一个含有功能性黄嘌呤 - 鸟嘌呤磷酸核糖转移酶（gpt）基因和两个串联排列的具有约400 bp序列同源性的G418抗性（新霉素，neo）基因片段的载体转染到CHO细胞中。从转染培养物中建立克隆细胞系，发现G418抗性回复子的自发频率在1×10⁻⁴至5×10⁻⁴之间。在其中两个细胞系中研究了烷化剂甲磺酸甲酯（MMS）和氮芥（HN2）诱导转染的neo基因重组的能力。用MMS处理使存活率降低至对照的10%后，这些细胞系中G418抗性回复子的频率呈剂量依赖性增加。用HN2处理后未观察到效果。所有G418抗性亚克隆都包含一个新的限制性片段，表明在截短的neo基因对中通过重排形成了一个完整的neo基因。因此，该系统可用于研究哺乳动物细胞中同源重组的分子机制和化学诱导性。
