PMID: 7546690May 1, 1995Paper

Construction of a cDNA library from human retinal pigment epithelial cells challenged with rod outer segments

Australian and New Zealand Journal of Ophthalmology
D M CavaneyI J Constable

Abstract

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. 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. 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 identi...Continue Reading

References

Apr 1, 1990·Biochemical Society Transactions·M CrossmanJ Mellerio
Dec 1, 1987·Experimental Eye Research·M O Hall, T Abrams
Jul 1, 1986·The Journal of Cell Biology·P L Mayerson, M O Hall
Jan 4, 1972·Biochemistry·P J O'BrienJ J Bungenberg de Jong
Jan 1, 1983·Survey of Ophthalmology·S Hayasaka

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Citations

Oct 9, 2002·The American Journal of Pathology·Piroska Elizabeth RakoczyChooi-May Lai

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