April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Generation of a Novel Optineurin Knockout Mouse
Author Affiliations & Notes
  • Frank S Ashburn
    Ophthalmology, Duke Eye Center, Durham, NC
  • Howard Bomze
    Ophthalmology, Duke Eye Center, Durham, NC
  • Celia McKee
    Ophthalmology, Duke Eye Center, Durham, NC
  • Ian Barak
    Ophthalmology, Duke Eye Center, Durham, NC
  • Henry Tseng
    Ophthalmology, Duke Eye Center, Durham, NC
  • Footnotes
    Commercial Relationships Frank Ashburn, None; Howard Bomze, None; Celia McKee, None; Ian Barak, None; Henry Tseng, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 5710. doi:
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      Frank S Ashburn, Howard Bomze, Celia McKee, Ian Barak, Henry Tseng; Generation of a Novel Optineurin Knockout Mouse. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5710.

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

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Abstract

Purpose: Mutations in the OPTN gene are associated with normal pressure glaucoma and amyotrophic lateral sclerosis (ALS), resulting in the selective loss of retinal ganglion cells and motor neurons, respectively. However, the pathophysiological mechanism of this process is poorly understood. We hypothesize that mutations lead to neuronal loss through a partial or complete “loss of function” mechanism. To test this hypothesis in vivo, we successfully generated a novel floxed optineurin mouse that can be utilized as a conditional knockout mouse and performed preliminary experiments to characterize retinal alterations.

Methods: LoxP sites have been introduced to flank the first coding exon of genomic mouse OPTN and the start codon, such that presence of the Cre recombinase would result in the excision of this DNA region. After backcrossing this mouse for multiple generations on a C57 background, we introduced Cre by breeding our floxed optineurin mice with a CMV-Cre mouse for ubiquitous Cre expression. Offsprings with heterozygous and homozygous genetic deletion of mouse optineurin were evaluated by utilizing qRT-PCR as well as Western immunoblotting. Retinal sections from these mice were evaluated with immunofluorescence staining for retinal markers such as Brn3, PKC-α, and calretinin.

Results: We successfully generated the floxed optineurin mouse as described above, which resulted in the genetic deletion of optineurin with CMV-Cre expression. While both heterozygous and homozygous knockout offsprings were obtained, the number of surviving homozygous optineurin knockout pups was lower than expected when compared to littermates with heterozygous optineurin knockout or no knockout. Western immunoblotting of isolated retinas showed that genetic deletion of mouse optineurin protein was found to be successful. Preliminary immunostaining in the retinas of our optineurin knockout mice showed thinning of the retina.

Conclusions: We successfully generated a floxed optineurin mouse that was utilized to genetically delete optineurin. In addition, we found that generalized loss of optineurin may alter viability in mice and lead to retinal changes. Future work will be required to fully characterize these findings. We anticipate that our conditional optineurin knockout mouse model will improve our functional understanding of optineurin in glaucoma and ALS, as well as provide an animal model to test candidate therapeutics.

Keywords: 533 gene/expression  
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