Yoon K, Cole-Strauss A, Kmiec E B
Department of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Proc Natl Acad Sci U S A. 1996 Mar 5;93(5):2071-6. doi: 10.1073/pnas.93.5.2071.
An experimental strategy to facilitate correction of single-base mutations of episomal targets in mammalian cells has been developed. The method utilizes a chimeric oligonucleotide composed of a contiguous stretch of RNA and DNA residues in a duplex conformation with double hairpin caps on the ends. The RNA/DNA sequence is designed to align with the sequence of the mutant locus and to contain the desired nucleotide change. Activity of the chimeric molecule in targeted correction was tested in a model system in which the aim was to correct a point mutation in the gene encoding the human liver/bone/kidney alkaline phosphatase. When the chimeric molecule was introduced into cells containing the mutant gene on an extrachromosomal plasmid, correction of the point mutation was accomplished with a frequency approaching 30%. These results extend the usefulness of the oligonucleotide-based gene targeting approaches by increasing specific targeting frequency. This strategy should enable the design of antiviral agents.
已开发出一种促进纠正哺乳动物细胞中游离型靶标单碱基突变的实验策略。该方法利用一种嵌合寡核苷酸，其由双链构象的连续RNA和DNA残基组成，两端带有双发夹帽。RNA/DNA序列设计为与突变位点的序列对齐，并包含所需的核苷酸变化。在一个模型系统中测试了嵌合分子在靶向校正中的活性，该模型系统旨在校正编码人肝/骨/肾碱性磷酸酶的基因中的点突变。当将嵌合分子导入含有位于染色体外质粒上的突变基因的细胞中时，点突变的校正频率接近30%。这些结果通过提高特异性靶向频率扩展了基于寡核苷酸的基因靶向方法的实用性。该策略应能实现抗病毒药物的设计。
