April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Correction of the Crb1rd8 allele and retinal phenotype in C57BL/6N mice via TALEN-mediated homology-directed repair
Author Affiliations & Notes
  • Mark P Krebs
    The Jackson Laboratory, Bar Harbor, ME
  • Benjamin E Low
    The Jackson Laboratory, Bar Harbor, ME
  • J. Keith Joung
    Molecular Pathology Unit, Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA
    Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA
  • Shengdar Q Tsai
    Department of Pathology, Harvard Medical School, Boston, MA
  • Patsy M Nishina
    The Jackson Laboratory, Bar Harbor, ME
  • Michael V Wiles
    The Jackson Laboratory, Bar Harbor, ME
  • Footnotes
    Commercial Relationships Mark Krebs, None; Benjamin Low, The Jackson Laboratory (P); J. Keith Joung, Transposagen Biopharmaceuticals (I); Shengdar Tsai, None; Patsy Nishina, None; Michael Wiles, The Jackson Laboratory (P)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 3281. doi:https://doi.org/
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Mark P Krebs, Benjamin E Low, J. Keith Joung, Shengdar Q Tsai, Patsy M Nishina, Michael V Wiles; Correction of the Crb1rd8 allele and retinal phenotype in C57BL/6N mice via TALEN-mediated homology-directed repair. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3281. doi: https://doi.org/.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose: To directly correct the mouse Crb1rd8 mutation, which is present in many inbred laboratory strains derived from C57BL/6N and complicates genetic studies of retinal disease in mice.

Methods: Fertilized C57BL/6NJ oocytes were coinjected with mRNAs encoding a transcription activator-like effector nuclease (TALEN) targeting the Crb1rd8 allele plus single-stranded oligonucleotides to correct the allele. The oligonucleotides included additional nucleotide changes to distinguish the corrected allele (Crb1em1Mvw) from wild-type Crb1 and to minimize TALEN re-cutting. Oligonucleotide length and the concentration of injected oligonucleotides and TALEN mRNAs were varied to optimize homology-directed repair of the locus. Following microinjection, embryos were carried to term in pseudopregnant females. Correction efficiency was assessed by PCR analysis of the Crb1em1Mvw allele. Phenotypic correction was demonstrated by fundus imaging and optical coherence tomography of live mice and by confocal fluorescence microscopy of retinal flatmounts.

Results: Under optimal conditions, homology-directed repair was observed in 27% (8/30) of live born animals and showed minimal illegitimate recombination of donor DNA. However, extensive founder mosaicism was evident, emphasizing the need to analyze offspring of founder animals. Unlike C57BL/6NJ mice, which exhibited external limiting membrane fragmentation and regional retinal dysplasia, heterozygous Crb1em1Mvw/Crb1rd8 mice showed a normal retinal phenotype.

Conclusions: The C57BL/6NJ Crb1rd8 mutation and its associated retinal phenotypes were efficiently corrected by TALEN-mediated homology-directed repair. The C57BL/6NJ-Crb1em1Mvw mice generated by this strategy will enhance ocular phenotyping efforts based on the C57BL/6N background, such as those implemented by the International Mouse Phenotyping Consortium (IMPC) project.

Keywords: 539 genetics • 696 retinal degenerations: hereditary • 538 gene transfer/gene therapy  
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×