Cloning of rod cGMP-phosphodiesterase α-subunit from normal dog and retinal degenerate Labrador Retriever

Purpose. The rod phosphodiesterase (PDE) α-subunit was examined using PCR to exclude PDE mutations as a cause for elevated cGMP and retinal degeneration in the Labrador Retriever. Methods. From retinas frozen in liquid nitrogen, custom cDNA libraries were made. Titers of the amplified Labrador Retriever and Beagle cDNA libraries were 5.9 × 10¹⁰ pfu/ml and 8.0 × 10⁹ respectively. Based on human retinal PDE α-subunit sequence, preliminary primer pairs were synthesized. One μl of each library was used as a template in 50μl PCRs, products were cloned and a minimum of three clones was sequenced. Based on these sequences new canine specific primers were made. Results. There was no difference in the nucleotide sequence between these two strains of dogs. The coding sequence of the rod-cone degenerate Labrador Retriever consists of 2586 basepairs, encoding an 862 amino acid polypeptide. The sequence is highly similar to it's human (96.9%), bovine (97.7%) and murine (97.2%) counterparts. Compared the these published rod sequences the canine sequence has two additional amino acids (P₈₄₇ and S₈₄₈). cGMP-binding sites and the catalytic domain are found as in the species mentioned. Conclusions. Further investigations are needed to find out the cause of the elevated cGMP levels in this mutant, now that we have excluded both catalytic subunits of cGMP-PDE.