Branched oligonucleotides induce in vivo gene conversion of a mutated EGFP reporter

Gene Ther. 2003 Oct;10(21):1830-40. doi: 10.1038/sj.gt.3302079.

Abstract

Branched oligonucleotides (b-oligonucleotides) based on a novel branching monomer were used for site-specific sequence alteration in vivo. With a stable integrated mutated enhanced green fluorescent protein (EGFP) template in Chinese hamster ovary cells, up to 0.1% EGFP-positive cells were counted after transfection with b-oligonucleotides. The presence of EGFP protein in converted cells was demonstrated by anti-EGFP immunocytochemistry. Genomic sequencing of converted cells showed in 40% of the analysed clones the corrected wild-type codon, while 9.3% of the sequences showed a corrected wild-type sequence and an additional collateral mutation. Despite the stable corrected genomic locus, converted cells entered selective apoptosis after 3-6 days. The cell line Irs-1 that is deficient in the homologous recombination pathway showed a reduced frequency of b-oligonucleotide-induced site-specific sequence conversion. The reduced conversion rates in the mutant cell line could be partly rescued by complementation with XRCC2 cDNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Apoptosis
  • Base Sequence
  • CHO Cells
  • Cricetinae
  • Female
  • Flow Cytometry
  • Gene Conversion
  • Gene Expression
  • Genes, Reporter*
  • Green Fluorescent Proteins
  • In Situ Nick-End Labeling
  • Luminescent Proteins / genetics*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed*
  • Transfection / methods*

Substances

  • Luminescent Proteins
  • Green Fluorescent Proteins