The overall goal of this research is to provide continually growing, non- tumorigenic cultures of specific retinal cell types. To achieve this end, normal Fisher 344 rat neuroretinal cells will be immortalized in vitro by retroviral-mediated gene transfer using the adenovirus E1A immortalizing gene. Infected cells which survive selection by G418/neomycin resistance will be cloned by limiting dilution to generate discrete clonal cell populations. These populations will be characterized by immunoreactivity to phenotype-specific markers which are available for all retinal cell types. The role of specific growth factors on retinal cell development can then be evaluated, while expression of retinal gene products induced by terminal mitosis and cell differentiation can be established. E1A- immortalized clonal populations will be exposed to three known modulators of cell growth and differentiation, namely retinoic acid/sodium butyrate, succinylated concanavalin a (SCA) and pigmented epithelium-derived factor (PEDF). Treated cells will be observed for morphological changes and/or alterations in phenotype. This will determine if E1A-immortalized cells can still be influenced by environmental diffusible factors. In addition, the expression of E1A and the cellular Rb gene/protein will be monitored by western and northern blot, as well immunocytochemistry for responses to these growth factors during growth inhibition and differentiation. Next, the histogenic capabilities of these individual cell lines will be assessed by rotation-mediated histotypic reaggregation, in which combinations of normal and E1A-immortalized cells are dissociated and allowed to reconstruct associations in culture. Integration in vitro can be assessed by light and electron microscopic analyses. Immortalized cells within reaggregates will be identified by virtue of E1A expression, or pre-labelling with tritiated thymidine. Information from these in vitro experiments will be used to select specific clonal populations for subretinal transplantation into histocompatible Fisher 344 rats to determine the potential tumorigenic or histogenic capabilities of these cells in vivo. These studies will provide the tools to study the effects of growth factors and environmental influences on retinal cell development, and lead to a better understanding of the effects of these factors on retinal disease.
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