Oligonucleotide Microarray Based Expression Analysis of the Cpfl1 Mutant - a Mouse Model of Cone and Cone-Rod Dystrophies

Abstract

Purpose:: The cpfl1 mutant (cone photoreceptor function loss 1) is a mouse model carrying mutations in the cone specific phosphodiesterase 6C (pde6c). The phenotype is characterized by a loss of cone photoreceptor function and a progressive degeneration of the cones. To investigate the biological events leading to the loss of photoreceptors we performed microarray experiments in two age stages.

Methods:: Retinae of cpfl1 and wildtype mice at the age of 4 and 8 weeks were dissected. RNA was isolated and 3 samples each hybridized on Affymetrix MOE 430 2.0 microarrays. Chip analysis was performed using the Array Assist 4.0 software (Stratagene) selecting all transcripts with a minimum change in expression level of 1.5 fold with a p-value less than 0.05 (t-Test without multiple testing correction). Gene regulation networks were generated by the Ingenuity Pathways Analysis 3.1 software. The study was performed in accordance with the ARVO Statement for the use of Animals in Ophthalmic and Visual Research.

Results:: 338 transcripts were differently regulated in the retinae of the 4 week old mice and 223 in those of the 8 week old animals. There was an overlap of 30 % between the two experiments. A large number of genes encoding proteins involved in phototransduction were down regulated. Gene regulation networks also revealed misregulations of genes asoiated with cell death, proliferation and gene expression. To verify the data 11 transcripts were analyzed by qRT-PCR using the LC 480 system (Roche) and could be entirely validated.

Conclusions:: The expression analysis of the cpfl1 mutant highlighted a clear misregulation of components of the phototransduction cascade in accordance with the loss of visual function that characterizes the phenotype. The cpfl1 mutant in general represents an appropriate animal model for the investigation of cone dystrophies in humans.

Keywords: gene microarray • gene/expression • genetics