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 (RPE) 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 contribute to the development of diseases due to a failure in outer segment phagocytosis and digestion by RPE cells. Here we demonstrate a way to study genes and their functions using a molecular biological approach and describing the first step involved in this process, the construction of a cDNA library.


RESULTS
Human RPE cells obtained from the eyes of a seven-year-old donor were cultured and challenged with bovine ROS. The culture was harvested and total RNA was extracted. Complementary DNA was transcribed from the messenger (m)RNA and was directionally cloned into the LambdaGEM-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.


CONCLUSIONS
Molecular biology and cell culture are important tools to be used in eye research, especially in areas where tissue is limiting and animal models are not available. We now have a ROS challenged RPE cDNA library which will be used to identify genes responsible for degrading phagocytosed debris within the retinal pigment epithelium.
Cavaney
D M
DM
Lions Eye Institute, Centre for Ophthalmology and Visual Science, University of Western Australia, Nedlands.
Rakoczy
P E
PE
Constable
I J
IJ
D002478:Cells, Cultured; D002648:Child; D018076:DNA, Complementary / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D010587:Phagocytosis / Q000502:physiology*; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000502:physiology*; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics; D012374:Rod Cell Outer Segment / Q000502:physiology*
D018076:DNA, Complementary; D012333:RNA, Messenger; D012313:RNA