July 2018
Volume 59, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2018
Assessment of cone outer segment defects and rescue in the Opn1mw/sw double knockout mouse
Author Affiliations & Notes
  • Tiffanie M. Dahl
    Ophthalmology & Visual Sciences, University of Utah, Salt Lake City, Utah, United States
  • Michelle Reed
    Ophthalmology & Visual Sciences, University of Utah, Salt Lake City, Utah, United States
  • Cecilia D. Gerstner
    Ophthalmology & Visual Sciences, University of Utah, Salt Lake City, Utah, United States
  • Guoxin Ying
    Ophthalmology & Visual Sciences, University of Utah, Salt Lake City, Utah, United States
  • Wentao Deng
    Ophthalmology, University of Florida, Gainesville, Florida, United States
  • William W Hauswirth
    Ophthalmology, University of Florida, Gainesville, Florida, United States
  • Wolfgang Baehr
    Ophthalmology & Visual Sciences, University of Utah, Salt Lake City, Utah, United States
  • Footnotes
    Commercial Relationships   Tiffanie Dahl, None; Michelle Reed, None; Cecilia Gerstner, None; Guoxin Ying, None; Wentao Deng, None; William Hauswirth, AGTC Inc. (C); Wolfgang Baehr, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 3054. doi:
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    • Get Citation

      Tiffanie M. Dahl, Michelle Reed, Cecilia D. Gerstner, Guoxin Ying, Wentao Deng, William W Hauswirth, Wolfgang Baehr; Assessment of cone outer segment defects and rescue in the Opn1mw/sw double knockout mouse. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3054.

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

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Abstract

Purpose : Knockout (KO) of either Opn1mw or Opn1sw in mouse results in either the dorsal or ventral retina no longer having detectable cone opsin expression. We seek to determine what effect the complete loss of cone opsins will have on cone outer segment structure and cone viability. We also seek to determine if cone structure and function can be rescued in the Opn1mw/sw double knockout (DKO) using gene therapy.

Methods : Opn1mw/sw DKO mice were generated by crossing Opn1mw KO with Opn1sw KO to create double heterozygotes (DHet) (males are Opn1mw-/y; Opn1sw+/-). DHets were then crossed to create the DKO. Cone function was assessed using electroretinography (ERG) Immunohistochemistry (IHC) was done using antibodies against M/L-opsin, S-opsin, mouse cone arrestin, and peanut agglutinin (PNA). Transmission electron microscopy (TEM) is done on glutaraldehyde fixed eyes. Rescue experiments are planned using AAV5 vector expressing M-opsin or S-opsin using subretinal injection.

Results : ERG showed Opn1mw/sw DKOs lack a photopic b-wave across all ages. IHC and confocal microscopy show that the outer segments of Opn1mw/sw DKO cones are absent, but based on cone arrestin staining cones remain viable. The cones of the Opn1mw/sw DKO have sufficient remaining structure that AAV mediated expression of M-opsin, S-opsin, or both opsins will rescue outer segment formation. Rescue experiments using AAV5 vector with the cone-specific PR2.1 or PR1.7 promoter driving M-opsin or S-opsin expression are currently being done using trans-corneal subretinal injection at P14. Rescue of cone function and structure will be assessed 6 weeks after injection using ERG, IHC, and TEM.

Conclusions : Cone opsins are required for outer segment formation, but not for cone viability. The Opn1mw/sw DKO mouse and rescue with AAV5-Opn1mw and AAV5-Opn1sw will provide a tool for further study of the mechanisms involved in cone ciliogenesis and trafficking of cone pigments to the outer segments.

Funding: T32EY024234; EY08123; EY019298; P30EY014800; Research to Prevent Blindness, New York, NY; Retina Research Foundation, Houston, TX

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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