Intracellular generation of single-stranded DNA for chromosomal triplex formation and induced recombination.
Intracellular generation of single-stranded DNA for chromosomal triplex formation and induced recombination.
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DOI：
10.1093/nar/29.24.5140
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发表时间：
2001-12
影响因子：
14.9
通讯作者：
H. J. Datta;Peter M. Glazer
中科院分区：
文献类型：
--
作者：
H. J. Datta;Peter M. Glazer
合成的三螺旋形成寡脱氧核糖核苷酸（TFO）已被用于改变基因表达和诱导细胞和动物中的靶向基因组修饰。然而，这种寡脱氧核糖核苷酸（ODN）的功效取决于有效的细胞内递送。测试了一种新型载体系统用于产生单链DNA（ssDNA）以在小鼠细胞中充当TFO。用一系列ssDNA载体转染携带可报告三链体刺激的染色体内重组的底物的小鼠细胞，并测定诱导的重组。用设计用于产生34 nt富含G的ssDNA的载体组转染，该ssDNA能够在报告底物内的30 bp多嘌呤靶位点处形成三链体，以196 x 10（-6）的频率产生重组体，而在模拟转染细胞中背景频率为45 x 10（-6）。当测试缺乏TFO序列插入物的载体组时或当单独转染组分载体时，未观察到诱导。经工程改造以表达富含C的34 nt序列（预期在生理条件下不形成三链体）的载体对背景没有影响。对来自转染细胞的裂解物的引物延伸分析证实了预期ssDNA的产生。这些结果表明，一个确定的序列的ssDNA分子可以产生细胞内使用一种新的载体系统，这种分子是积极的介导三链体依赖的染色体事件。在细胞内产生活性TFO的能力可能为基于三链体的基因靶向策略提供新的基础。
Synthetic triple helix-forming oligodeoxyribonucleotides (TFOs) have been used to alter gene expression and to induce targeted genome modification in cells and animals. However, the efficacy of such oligodeoxyribonucleotides (ODNs) depends on efficient intracellular delivery. A novel vector system was tested for the production of single-stranded DNA (ssDNA) to serve as a TFO in mouse cells. Mouse cells carrying a substrate that can report triplex-stimulated intrachromosomal recombination were transfected with a series of ssDNA vectors, and induced recombination was assayed. Transfection with a vector set designed to generate a 34 nt G-rich ssDNA capable of triplex formation at a 30 bp polypurine target site within the reporter substrate yielded recombinants at a frequency of 196 x 10(-6), versus a background frequency of 45 x 10(-6) in mock transfected cells. No induction was seen when a vector set lacking the TFO sequence insert was tested or when the component vectors were transfected individually. Vectors engineered to express a C-rich 34 nt sequence (not expected to form triplex under physiological conditions) had no effect over background. Primer extension analyses on lysates from transfected cells confirmed the production of the intended ssDNAs. These results suggest that ssDNA molecules of a defined sequence can be generated intracellularly using a novel vector system and that such molecules are active in mediating triplex-dependent chromosomal events. The ability to produce active TFOs within cells may provide a new foundation for triplex-based gene targeting strategies.