Targeted gene correction by small single-stranded oligonucleotides in mammalian cells
Targeted gene correction by small single-stranded oligonucleotides in mammalian cells
复制标题
DOI：
10.1038/sj.gt.3301414
复制
发表时间：
2001-03-01
期刊：
影响因子：
5.1
通讯作者：
Yoon, K
中科院分区：
文献类型：
--
作者：
Igoucheva, O;Alexeev, V;Yoon, K
我们证明，相对较短的单链寡核苷酸，除了与目标碱基的单一错配外，与目标序列同源的25-61个碱基，能够纠正突变的β-半乳糖苷酶基因中的单点突变(G到A)，在哺乳动物细胞的核提取液、表皮体和染色体中，纠正率分别约为0.05%、1%和0.1%。令人惊讶的是，这些短单链寡核苷酸(ODN)显示出与使用相同系统测量的嵌合RNA-DNA寡核苷酸相似的基因校正频率。核抽提物中ODN诱导的体外基因校正不依赖于寡核苷酸的长度或极性。相反，上体和染色体的基因校正高度依赖于ODN的长度和极性。同源性为45个核苷酸的ODN出现频率最高，反义取向的ODN出现频率是正义取向的1000倍，这可能是转录对基因校正的影响。与相同序列的核糖体寡核苷酸相比，脱氧寡核苷酸具有更高的基因校正频率。这些结果表明，相对较短的ODN可以在哺乳动物细胞中以与嵌合RNA-DNA寡核苷酸相似的频率对靶序列进行序列特异性改变。
We demonstrate that relatively short single-stranded oligodeoxynucleotides, 25-61 bases homologous to the target sequence except for a single mismatch to the targeted base, are capable of correcting a single point mutation (G to A) in the mutant beta -galactosidase gene, in nuclear extracts, episome, and chromosome of mammalian cells, with correction rates of approximately 0.05%, 1% and 0.1%, respectively. Surprisingly, these short single-stranded oligonucleotides (ODN) showed a similar gene correction frequency to chimeric RNA-DNA oligonucleotide, measured using the same system. The in vitro gene correction induced by ODN in nuclear extracts was not dependent on the length or polarity of the oligonucleotide. In contrast, the episomal and chromosomal gene corrections were highly dependent on the ODN length and polarity. ODN with a homology of 45 nucleotides showed the highest frequency and ODN with antisense orientation showed a 1000-fold higher frequency than sense orientation, indicating a possible influence of transcription on gene correction. Deoxyoligonucleotides showed a higher frequency of gene correction than ribo-oligonucleotides of the identical sequence. These results show that a relatively short ODN can make a sequence-specific change in the target sequence in mammalian cells, at a similar frequency as the chimeric RNA-DNA oligonucleotide.