Validation of oligonucleotide-mediated gene editing

Gene Ther. 2009 Jun;16(6):824-6. doi: 10.1038/gt.2009.32. Epub 2009 Apr 2.

Abstract

Several independent groups have reported targeted genomic editing in mammalian cells mediated by synthetic oligonucleotides. Nevertheless, the validity of data has been disputed because of experimental artefacts, inconsistent findings and low reproducibility. Here, we describe experiments designed to meet stringent criteria and completely eliminate artefactual results. In particular, by targeting cells expressing mutated enhanced green fluorescence protein (EGFP), which allow editing measurements at the protein level, and analyzing corrected clones by Southern blotting, we rigorously excluded spontaneous reversion, contamination artefacts, false-positives, or overestimation. Our findings provide unequivocal authentication that oligonucleotide-mediated gene editing is a real, not artefactual, phenomenon--a vital starting point from which to develop the technology into practical applications.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Animals
  • Artifacts*
  • Base Sequence / genetics
  • Blotting, Southern
  • CHO Cells
  • Clone Cells
  • Cricetinae
  • Cricetulus
  • Gene Targeting / methods
  • Green Fluorescent Proteins / genetics*
  • Oligonucleotides / genetics*
  • Oligonucleotides / pharmacology
  • Reproducibility of Results
  • Targeted Gene Repair / methods*

Substances

  • Oligonucleotides
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins