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.
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Acknowledgements
We thank Stefan Krauss and Petter A Olsen for the CHO-mEGFP cells and Rafael J Yáñez for helpful comments. This work was supported by a MRC PhD studentship to PD.
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Disterer, P., Simons, J. & Owen, J. Validation of oligonucleotide-mediated gene editing. Gene Ther 16, 824–826 (2009). https://doi.org/10.1038/gt.2009.32
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DOI: https://doi.org/10.1038/gt.2009.32
