Conditionally immortalized TM cell and RGC lines from Animal Model

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Abstract Glaucoma is a leading cause of irreversible vision loss and blindness in the world. Two major sites of glaucoma damage are the trabecular meshwork (TM), which leads to elevated intraocular pressure, and retinal ganglion cells (RGCs) that progressively die in glaucoma, leading to the loss of vision associated with this disease. A great deal has been learned about the cell and molecular biology of glaucoma through research on cultured TM cells and RGCs. However, there are significant limitations with current TM cell and RGC cultures. TM cells grow relatively slowly and rapidly senesce, limiting studies to relatively small cell numbers of early cell passages. RGCs are terminally differentiated, and therefore, primary RGCs must be prepared from neonatal rodent eyes for each experiment conducted. Although immortalized cell lines have been generated, these cells continuously proliferate in culture and have lost other phenotypic characteristics of primary TM cells and RGCs. There is a definite need for new TM cell and RGC lines that can be easily propagated and still retain the phenotypic characteristics of primary cell cultures. The ImmortoMouse was developed as a unique resource to generate conditionally immortalized cells from a wide variety of tissues that will continuously proliferate when the cells are cultured at permissive temperatures, but regain their normal phenotypes when grown at non-permissive temperatures. Our hypothesis is that conditionally immortalized mouse RGC and TM cell lines from the ImmortoMouse can be isolated, which will have the characteristics of primary TM cells and RGCs when grown under non-permissive conditions. The following Specific Aims will address this hypothesis. (SA#1) We will develop and characterize conditionally immortalized mouse TM cell lines that will rapidly proliferate under permissive conditions (culture at 33oC with ¿IFN) and will have characteristics of primary TM cells when grown under non-permissive conditions (culture at 39oC without ¿IFN). (SA#2) We will develop and characterize conditionally immortalized mouse RGC lines that will rapidly proliferate under permissive conditions and will have characteristics of primary RGCs when grown under non- permissive conditions. This project is very innovative in several ways. (A) We will isolate and characterize the first conditionally immortalized TM and RGC cell lines. (B) We also have developed a novel method using phagocytized magnetic microspheres to purify mouse TM cells. These lines will be made available to a wide variety of vision researchers to: (a) better understand TM and RGC biology, (b) discover and characterize new pathogenic pathways, and (c) test potential new therapeutic agents. !