The Q64Ter Mouse Model for Studying the Repair of Mutations in Neurons

Abstract

Purpose:: Retinitis Pigmentosa (RP) comprises a group of inherited degenerative disorders of the retina, affecting approximately 1.5 million people worldwide. The autosomal dominant form of RP is most often triggered by point mutations in the rhodopsin gene. We have developed mouse models with disease-causing mutations in the rhodopsin gene for the dual purpose of studying DNA repair processes in terminally differentiated neurons and evaluating novel therapeutics for dominant genetic disorders. We previously developed and characterized a mouse model that carries a fusion of the normal human rhodopsin gene and GFP in place of the endogenous mouse rhodopsin gene.

Methods:: We used homologous recombination in embryonic stem cells to generate a knock-in mouse line that carries a Q64ter mutation that prevents expression of rhodopsin-GFP to serve as exquisitely sensitive reporters of gene correction. Correction of the mutation in a rod photoreceptor will activate rhodopsin-GFP expression, turning the rod outer segment bright green in an otherwise black background. We are investigating a variety of therapeutic techniques for recovering rhodopsin activity, including read-through drugs and the use of custom nucleases to target the repair of mutations. The retinal degeneration caused by the Q64ter mutation is being studied using cryosectioning, immunohistochemistry and electroretinography (ERG).

Results:: Heterozygote mice with Q64ter rhodopsin-GFP have been characterized and show no evidence of mislocalization of rhodopsin by immunohistochemistry, then begin to show early degeneration, detected by measuring the size of the outer nuclear layer, at three months of age. Electophysiological studies show these three-month old mice have significantly decreased a-waves (p<0.05) compared to wild-type littermates. Also, recovery of the a-wave using a double-flash protocol indicates that the Q64ter rhodopsin-GFP heterozygote mice recovered significantly faster compared with WT and resembled the recovery seen in the rhodopsin null heterozygote mice.

Conclusions:: The Q64ter rhodopsin-GFP mice have slow degeneration, making these mice an optimal system for evaluating gene therapies in order to detect repair in neurons.

Keywords: gene transfer/gene therapy • transgenics/knock-outs • retina