Hu Yiling, Parekh-Olmedo Hetal, Drury Miya, Skogen Michael, Kmiec Eric B
Department of Neuroscience, Dalhousie University, Halifax, Nova Scotia.
Mol Biotechnol. 2005 Mar;29(3):197-210. doi: 10.1385/MB:29:3:197.
Targeted gene repair uses short DNA oligonucleotides to direct a nucleotide exchange reaction at a designated site in a mammalian chromosome. The widespread use of this technique has been hampered by the inability of workers to achieve robust levels of correction. Here, we present a mammalian cell system in which DLD-1 cells bearing integrated copies of a mutant eGFP gene are repaired by modified single-stranded DNA oligonucleotides. We demonstrate that two independent clonal isolates, which are transcribed at different levels, are corrected at different frequencies. We confirm the evidence of a strand bias observed previously in other systems, wherein an oligonucleotide designed to be complementary to the nontranscribed strand of the target directs a higher level of repair than one targeting the transcribed strand. Higher concentrations of cell oligonucleotides in the electroporation mixture lead to higher levels of correction. When the target cell population is synchronized into S phase then released before electroporation, the correction efficiency is increased within the entire population. This model system could be useful for pharmacogenomic applications of targeted gene repair including the creation of cell lines containing single-base alterations.
靶向基因修复利用短DNA寡核苷酸在哺乳动物染色体的指定位点引导核苷酸交换反应。该技术的广泛应用因研究人员无法实现高效的校正水平而受到阻碍。在此，我们展示了一种哺乳动物细胞系统，其中携带突变型eGFP基因整合拷贝的DLD-1细胞通过修饰的单链DNA寡核苷酸进行修复。我们证明，两个转录水平不同的独立克隆分离株，其校正频率也不同。我们证实了先前在其他系统中观察到的链偏好证据，即设计与靶标非转录链互补的寡核苷酸比靶向转录链的寡核苷酸能引导更高水平的修复。电穿孔混合物中更高浓度的细胞寡核苷酸会导致更高水平的校正。当靶细胞群体同步进入S期然后在电穿孔前释放时，整个群体中的校正效率会提高。该模型系统可用于靶向基因修复的药物基因组学应用，包括创建包含单碱基改变的细胞系。
