Characterization of Mertk Mutation and Development of A Polymerase Chain Reaction Genotyping Method in Royal College...

The retinal pigment epithelium (RPE) cells are a single layer of cells with specific functions in vision. The Mertk gene, encoding a receptor tyrosine kinase, is critical for the phagocytic function of retinal RPE cells. Mutations in Mertk disrupt RPE function and contribute to retinal degeneration. This study aims to characterize the Mertk mutation in Royal College of Surgeons (RCS) rats and develop a polymerase chain reaction (PCR)-based method for genotyping these mutations to improve colony management. DNA was extracted from mutant Mertk^-/- and wild-type rats, followed by PCR amplification using primers flanking the deletion region. Sequencing of the PCR products was performed to identify the precise nature of the mutation. A PCR-based genotyping method was then developed to distinguish between homozygous and heterozygous mutants. Sequencing revealed a 1850 bp deletion in the Mertk gene, resulting in a truncated protein that potentially impairs RPE phagocytosis. The newly developed PCR method successfully differentiated between homozygous and heterozygous mutant rats. This genotyping technique proved to be efficient and reliable, facilitating the management of rat colonies for research purposes. This study provides a detailed molecular characterization of the Mertk mutation in RCS rats, enhancing our understanding of Mertk-related retinal degenerative diseases. The development of a robust PCR-based genotyping method enables efficient differentiation of rat genotypes, aiding in the creation and maintenance of rat models for future research. These findings underscore the importance of molecular characterization in advancing our understanding of genetic models and improving research meth odologies.