Pierce E A, Liu Q, Igoucheva O, Omarrudin R, Ma H, Diamond S L, Yoon K
FM Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Gene Ther. 2003 Jan;10(1):24-33. doi: 10.1038/sj.gt.3301857.
We have investigated the use of single-stranded oligodeoxy-nucleotides (ssODN) to produce specific single-base alterations in episomal and chromosomal DNA in mouse embryonic stem (ES) cells. Two different reporter genes, EGFP and LacZ, each with a single point mutation that inactivates reporter activity, were used. ssODN homologous to the target sequence, except for a single mismatch at the mutant base, were used to correct the mutant reporter genes. When tested in CHO-K1 cells, the ssODN showed correction rates of 0.5-1.0%, consistent with prior reports. ssODN in the antisense orientation provided higher rates of gene conversion than those in the sense orientation for both reporter genes. Nuclear extracts from mouse ES cells exhibited nearly the same correction activity as extracts from CHO-K1 cells. ssODN corrected the mutant bases of both episomal and chromosomal mutant reporter genes in mouse ES cells. Although the efficiency of gene correction observed in ES cells is low, approximately 10(-4), these results demonstrate that ssODN can produce single-base alterations in the genomic DNA of mouse ES cells. As conversion efficiency is improved by the continued development of oligonucleotide structure and DNA delivery methods, ssODN could be used to produce ES cells with specific mutations in any gene in a single step. The targeted ES cells could in turn be used to create accurate mouse models of inherited diseases.
我们研究了使用单链寡脱氧核苷酸（ssODN）在小鼠胚胎干细胞（ES细胞）的游离型和染色体DNA中产生特定单碱基改变的情况。使用了两种不同的报告基因，EGFP和LacZ，每个报告基因都有一个使报告基因活性失活的单点突变。与靶序列同源的ssODN，除了在突变碱基处有一个单碱基错配外，用于校正突变的报告基因。在CHO-K1细胞中进行测试时，ssODN的校正率为0.5-1.0%，与先前的报告一致。对于这两个报告基因，反义方向的ssODN比正义方向的ssODN提供更高的基因转换率。小鼠ES细胞的核提取物表现出与CHO-K1细胞提取物几乎相同的校正活性。ssODN校正了小鼠ES细胞中游离型和染色体突变报告基因的突变碱基。尽管在ES细胞中观察到的基因校正效率很低，约为10^(-4)，但这些结果表明ssODN可以在小鼠ES细胞的基因组DNA中产生单碱基改变。随着寡核苷酸结构和DNA递送方法的不断发展提高了转换效率，ssODN可用于一步产生任何基因具有特定突变的ES细胞。这些经过靶向修饰的ES细胞进而可用于创建遗传性疾病的精确小鼠模型。
