Targeted gene correction of episomal DNA in mammalian cells mediated by a chimeric RNA center dot DNA oligonucleotide

被引:199
作者
Yoon, K [1 ]
ColeStrauss, A [1 ]
Kmiec, EB [1 ]
机构
[1] THOMAS JEFFERSON UNIV,JEFFERSON CANC INST,DEPT PHARMACOL,PHILADELPHIA,PA 19107
关键词
D O I
10.1073/pnas.93.5.2071
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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.
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页码:2071 / 2076
页数:6
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