CONSTRUCTION OF A CDNA LIBRARY FROM HUMAN RETINAL-PIGMENT EPITHELIAL-CELLS CHALLENGED WITH ROD OUTER SEGMENTS

Background: To study genes expressed by retinal pigment epithelial (RP E) cells during phagocytosis and digestion of rod outer segments (ROS) , a complementary (c)DNA library was produced using an in-vitro model. The cDNA library can be used to study molecular changes which contrib ute to the development of diseases due to a failure in outer segment p hagocytosis and digestion by RPE cells. Here we demonstrate a way to s tudy genes and their functions using a molecular biological approach a nd describing the first step involved in this process, the constructio n of a cDNA library. Methods and results: Human RPE cells obtained fro m the eyes of a seven-year-old donor were cultured and challenged with bovine ROS. The culture was harvested and total RNA was extracted. Co mplementary DNA was transcribed from the messenger (m)RNA and was dire ctionally cloned into the LambdGEM-4 bacteriophage vector successfully . Some clones were picked and the DNA extracted, to determine the size of the inserts as a measure of the quality of the library. Conclusion s: Molecular biology and cell culture are important tools to be used i n eye research, especially in areas where tissue is limiting and anima l models are not available. We now have a ROS challenged RPE cDNA libr ary which will be used to identify genes responsible for degrading pha gocytosed debris within the retinal pigment epithelium.