The overall goal of this research is to provide specific cultures of actively growing, transformed retinal cells using the chick neuroretinal-retroviral system. Use of temperature sensitive and transformation defective oncogenes will permit the manipulation of specific retinal gene products and will establish an in vitro model system for a better understanding of retinal disorders. Embryonic neural retinal cells will be enriched by flow cytometry-cell sorting for infection of retinal subpopulations while transformed cells will be cloned from actively growing, heterogeneous populations by cell sorting and limiting dilution. Retinal cultures will be characterized using a battery of specific retinal monoclonal antibodies and also electronmicroscopy. Moreover, the presence of differentiation markers such as nerve specific enolase, neurofilament protein, neurotransmitters and surface glycoconjugates will be examined by immunocytochemistry and high performance liquid chromotography. In particular, the expression of the protein subunits of transducin, found to change following oncogene transformation, will be manipulated with cyclic nucleotides and neurotransmitters, while the cell type responsible for transducin Beta-like polypeptide enhancement will be identified using specific antisera. Transformed and actively growing retinal cells will also be placed in a three dimensional reaggregated culture system to establish if important cell-cell associations can be formed. Electronmicroscopy and monoclonal antibodies will reveal specific cell types while steroid and cyclic nucleotide treatments of transformed aggregated cultures will establish if important recognition events can be induced. These retinal culture systems will prove valuable in our understanding of the factors influencing normal development as well as the diseased state.
