July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
withdrawn - Normal Retinal Phenotype in the Aged Global Cas9 Expressing Mouse Strain
Author Affiliations & Notes
  • Mark Ellsworth Kleinman
    Ophthalmology & Visual Sci, Univ of Kentucky, Lexington, Kentucky, United States
  • Kyung Sik Jung
    Ophthalmology & Visual Sci, Univ of Kentucky, Lexington, Kentucky, United States
  • Sushil Dubey
    Ophthalmology & Visual Sci, Univ of Kentucky, Lexington, Kentucky, United States
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 386. doi:
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      Mark Ellsworth Kleinman, Kyung Sik Jung, Sushil Dubey; withdrawn - Normal Retinal Phenotype in the Aged Global Cas9 Expressing Mouse Strain. Invest. Ophthalmol. Vis. Sci. 2018;59(9):386.

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

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Abstract

Purpose : The CRISPR-Cas9 system has emerged as powerful and precise genetic editing tool to create mouse models of human disease. To develop such models, several approaches are being evaluated including the use of a global expressing Cas9 C57/BL6J strain that can be used with tissue targeted guide RNA expressing vectors. A commonly used strain (Rosa26-Cas9 knockin, Jax Mice) constitutively and globally expresses Cas9, a FLAG epitope tag and EGFP driven by a CAG promoter. In this study, we build on previous studies showing safety and efficacy of Cas9 expression in the mouse RPE and perform additional studies analyzing retinal phenotype and function at 6-12 months of age.

Methods : Globally expressing Cas9 mice (age 6-12 months, n=6-8) were evaluated for retinal degeneration by color fundoscopy, SD-OCT (Spectralis), full-field flash ERG (Diagnosys) and ZO-1 immunofluorescence (Abcam) on flatmounts. Age matched Wild-type mice (C57BL/6J) served as controls. Statistical tests used included Mann-Whitney U, ANOVA, and Fisher's exact. Genotyping for the commonly found rd8 mutation was performed (n=3, independent Cas9 knockin mice) with PCR and Sanger sequencing.

Results : Retinal phenotype of the globally expressing Cas9 appeared normal at 6-12 months of age by color fundoscopy, SD-OCT, and ZO-1 staining as compared to Wild-type controls (p>0.05). In similar studies, retinal function as tested with full-field ERG showed normal scotopic responses (p>0.05). Rd8 mutation analyses demonstrated normal Wild-type alleles.

Conclusions : Our previous studies suggest that CRISPR-Cas9 gene editing is well-tolerated by retinal tissues and does not induce cytotoxicity or degeneration in mice 6-8 weeks of age. Here, we show that the global constitutively expressing Cas9 knockin mouse has normal retinal phenotype and function out to 12 months of age without any evidence of an rd8 mutation. This mouse strain is a useful tool for performing in vivo targeted gene-editing of the retinal tissues to develop novel disease models.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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