Targeted gene correction of episomal DNA in mammalian cells mediated by a chimeric RNA.DNA oligonucleotide.
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.
Yoon
K
K
Department of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Cole-Strauss
A
A
Kmiec
E B
EB
D000469:Alkaline Phosphatase / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D005818:Genetic Engineering / Q000379:methods*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000737:chemistry*; D010957:Plasmids; D017354:Point Mutation; D014162:Transfection
D009841:Oligonucleotides; D000469:Alkaline Phosphatase