The long aboutterm goal of this project is to define all the proteins expressed in rodent retinas in order to provide vision researchers with information regarding the proteins actually expressed in retinal cells. Protein analysis by the combination of two-dimensional (2-D) gel electrophoresis, mass spectrometry, and genome database search has impacted on the progress of biomedical sciences. This new breed of technology is generally called """"""""proteomics"""""""" and enables massive analysis of proteins extracted from cells or tissues. In the past two decades the understanding of visual transduction pathway and its underlying molecular mechanisms has substantially been improved by the use of rod outer segment preparations from various animal models including bovine, frog, and other animals. The recent transgenic mouse model also has substantiated and extended much of the previous knowledge on the molecular mechanisms underlying vision. In this proposal, a proteomic approach will be used to characterize the proteins expressed in the retinas of laboratory rodents, rats and mice. The reasons for the choice of rodents as a model to study retinal protein expression are three-fold. First is the similarity in gene sequence between human and rodents. Second is the availability of transgenic animal models and the accumulated literatures on the physiology and pathological changes of rodent retinas induced by environmental or genetic perturbation. Finally, the lower costs and ease of experimental manipulation makes a rodent model practical. There are three specific aims for this proposal.
Aim 1 is to profile virtually all the retinal proteins that are extracted from rodent retinas and separated on 2-D gels. Protein profiling will be made along the developmental time axis by investigating retinas at different postnatal days. In this aim, efforts will also be made to develop a technique to remove abundant proteins from the protein extract in order to investigate proteins expressed in minor quantities, and also to develop a mass spectrometric technique to quantify proteins by stable isotope labeling.
Aim 2 is to profile immunohistochemical localization of selected proteins identified in Aim 1. In this aim, a defined set of criteria will be used so that the proteins chosen will be classified into groups that are physiologically distinct.
Aim 3 is to make the proteomic information accessible on a web-based database so that vision researchers can retrieve data when needed. Accomplishment of this project will help researchers investigating vision and its disorders at the protein level using rodent models.
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