Generation and Analysis of Canine Retinal ESTs: Isolation and Expression of Retina-Specific Gene Transcripts

Abstract

Canine generalized progressive retinal atrophies (gPRA) are a group of degenerative retinal diseases that are a major cause of hereditary blindness in a number of dog breeds. The expressed sequence tag (EST) approach was used to identify and characterize potential candidate genes from canine retinal cDNA libraries. Both conventional and subtractive canine retinal cDNA libraries were constructed and analyzed. Differential hybridization was performed to identify abundantly retinal expressed cDNA clones. Sequences of both random and abundantly expressed clones were analyzed using GCG software and searched against GenEMBL databases. For genes of interest isolated from the libraries, Northern blotting and RT-PCR were performed to determine mRNA expression of the genes. DNA sequences from 85 differentially expressed clones and 100 random cDNAs were obtained and analyzed. A higher percentage of abundantly retina-expressed clones showed homology to database sequences compared with random clones (72 versus 43%). Five retinal genes and 2 anonymous retinal ESTs were selected to analyze mRNA expression. The five known genes, namely HRG4/unc119, cGMP-PDEA, transducin 1A, opsin, and sFRP2 showed retina-specific expression. In anonymous ESTs, clone p81 revealed retina-specific expression, while p3 showed expression in each of 14 canine tissues. Transcripts of the canine secreted frizzled related protein 2 (sFRP2) gene showed surprisingly high abundance in the canine retina. The isolated retinal ESTs here will be useful resources for further investigation of canine retinal function and canine genome mapping.