DNA oligonucleotides and plasmids perform equally as donors for targeted gene conversion.
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.
Wagner
Stefan
S
Ruakura Research Centre, Hamilton, New Zealand. stefan.wagner@agresearch.co.nz
McCracken
Judi
J
Cole
Sally
S
Laible
Götz
G
D000818:Animals; D001483:Base Sequence; D002461:Cell Line, Transformed; D006224:Cricetinae; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004260:DNA Repair; D005785:Gene Conversion; D018390:Gene Targeting; D015316:Genetic Therapy / Q000379:methods; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009841:Oligonucleotides / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000627:therapeutic use; D010957:Plasmids / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism / Q000627:therapeutic use; D017354:Point Mutation; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D009841:Oligonucleotides; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D004247:DNA