Construction of a G-90-D rhodopsin mouse model

M. L. Fowler, R. A. Bush, D. K. Gerweck, K. Scott, J. R. Richards, P. A. Sieving

Research output: Contribution to journalArticlepeer-review

Abstract

Purpose. The G-90-D rhodopsin mutation in humans produces night blindness with minimal retinal degeneration. The mechanism is believed to involve constitutive activation due to charge redistribution in the opsin pocket by the addition of a negative aspartate residue across from the protonated Schiff base at the retinal attaachment site. A mouse model was sought to examine the question further. Methods. A 15 kb rhodopsin mouse genomic clone containing 5 kb of the promoter region was isolated. The G-90-D mutation was inserted by site-directed mutagenesis. An A-337-V change was introduced into one of the constructs as the epitope site for the 3A6 antibody (gift of R. Molday). The constructs were injected into C57/BL6 x DBA embryos, and 22 founder lines resulted. Founders were examined for retinal function by flash ERG. Lines with elevated ERG thresholds were examined histologically for degeneration, Immunohistochemistry was used to determine retinal distribution of mutant rhodopsin. Results. Of 11 lines examined thus far, 3 show elevated B-wave thresholds. In one line, 4-6 week old animals had ERG thresholds elevated 3 log units, similar to the human patients, but also showed thinning of the ONL to 4-6 nuclei; by three months, no ERG activity remained. However, one F2 animal in this line at 6 weeks is showing the 3 log elevation but retains a 120uV b-wave at maximal intensity and may mimic the human phenotype, although histology remains to be done. Conclusion. We have not yet observed a G-90-D mouse line with a clear functional deficiency in the absence of retinal degeneration. However, based on preliminary characterization, one line appears promising to provide a suitable model for the equivalent human mutation.

Original languageEnglish (US)
Pages (from-to)S698
JournalInvestigative Ophthalmology and Visual Science
Volume37
Issue number3
StatePublished - Feb 15 1996
Externally publishedYes

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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