Hellgren D, Lambert B
Karolinska Institute, Department of Clinical Genetics, Karolinska Hospital, Stockholm, Sweden.
Mutat Res. 1989 Dec;215(2):197-204. doi: 10.1016/0027-5107(89)90184-x.
Possible mechanisms for homologous recombination in CHO cells have been investigated using a stably integrated vector, pIII-14gpt. The vector contains 2 inactive neo gene fragments in tandem arrangement. Functional neo gene activity can be restored by recombination between homologous regions in the 2 fragments. Cells in which this event has taken place become resistant to the antibiotic G418. Possible mechanisms for neo gene reactivation in this system are unequal exchange between chromatids, intrachromatidal deletion and gene conversion. DNA from a total of 74 G418-resistant cell clones have been isolated, and analyzed on Southern blots using neo-specific probes. Rearrangements of neo-specific restriction fragments were found to have occurred in all cell clones. In 50% of the revertants, these rearrangements can be explained by a deletion which brings the complementary regions in the 2 neo gene fragments together. One single revertant (1.3%) shows a possible gene conversion event. The other isolated revertants (about 48%) contain more complex rearrangements. These results indicate that the predominating recombination mechanism for reactivation of the neo gene in this system is either a deletion within a chromatid or an unequal exchange between sister chromatids.
利用稳定整合载体pIII - 14gpt对中国仓鼠卵巢（CHO）细胞中同源重组的可能机制进行了研究。该载体包含两个串联排列的无活性新霉素（neo）基因片段。通过两个片段中同源区域之间的重组可恢复功能性neo基因活性。发生了此事件的细胞对抗生素G418产生抗性。该系统中neo基因重新激活的可能机制是染色单体之间的不等交换、染色体内缺失和基因转换。已从总共74个G418抗性细胞克隆中分离出DNA，并使用neo特异性探针在Southern印迹上进行分析。发现在所有细胞克隆中都发生了neo特异性限制性片段的重排。在50%的回复突变体中，这些重排可以用一种缺失来解释，这种缺失使两个neo基因片段中的互补区域聚集在一起。一个单一的回复突变体（1.3%）显示出可能的基因转换事件。其他分离出的回复突变体（约48%）包含更复杂的重排。这些结果表明，该系统中neo基因重新激活的主要重组机制要么是染色体内的缺失，要么是姐妹染色单体之间的不等交换。
