Abstract
Site-specific gene modifications in cells are initiated by the introduction of exogenous DNA. We used a recently established cell assay to compare the ability of DNA donors to induce a single point mutation that converts a target gene encoding blue fluorescent protein (BFP) into expressing green fluorescent protein (GFP). In a chromosomal assay with cells stably expressing BFP, we showed that fluorescently labeled single-stranded oligonucleotides and a donor plasmid cotranscribing a red fluorescent protein provide similar efficiencies in triggering BFP–GFP conversions. In transient cotransfections, an isogenic donor plasmid comprising a nonfunctional GFP gene yielded a greater efficiency for the conversion of the BFP target gene than a nonisogenic donor, and all plasmid donors were superior to oligonucleotides.
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Acknowledgments
We are grateful to Peter Beetham and Greg Gocal (Cibus LLC, San Diego, CA) for providing us with fluorescently labeled oligonucleotides and Jeff Sommer (North Carolina State University, Raleigh, NC) for supplying us with CHO-BFP cell lines. We would also like to thank an anonymous reviewer for valuable experimental advice. This study was funded by the New Zealand Foundation for Research and Technology.
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Wagner, S., McCracken, J., Cole, S. et al. DNA Oligonucleotides and Plasmids Perform Equally as Donors for Targeted Gene Conversion. Biochem Genet 48, 897–908 (2010). https://doi.org/10.1007/s10528-010-9370-z
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DOI: https://doi.org/10.1007/s10528-010-9370-z