ZFIN ID: ZDB-PUB-170401-3
PCR artifact in testing for homologous recombination in genomic editing in zebrafish
Won, M., Dawid, I.B.
Date: 2017
Source: PLoS One   12: e0172802 (Journal)
Registered Authors: Dawid, Igor B., Won, Minho
Keywords: Homologous recombination, Polymerase chain reaction, Zebrafish, Genetic loci, Embryos, Recombination reactions, Southern blot, Nucleotide sequencing
MeSH Terms:
  • Animals
  • Artifacts
  • Blotting, Southern
  • Gene Editing
  • Homologous Recombination/genetics
  • Polymerase Chain Reaction
  • Zebrafish/genetics*
PubMed: 28362803 Full text @ PLoS One
ABSTRACT
We report a PCR-induced artifact in testing for homologous recombination in zebrafish. We attempted to replace the lnx2a gene with a donor cassette, mediated by a TALEN induced double stranded cut. The donor construct was flanked with homology arms of about 1 kb at the 5' and 3' ends. Injected embryos (G0) were raised and outcrossed to wild type fish. A fraction of the progeny appeared to have undergone the desired homologous recombination, as tested by PCR using primer pairs extending from genomic DNA outside the homology region to a site within the donor cassette. However, Southern blots revealed that no recombination had taken place. We conclude that recombination happened during PCR in vitro between the donor integrated elsewhere in the genome and the lnx2a locus. We conclude that PCR alone may be insufficient to verify homologous recombination in genome editing experiments in zebrafish.
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