? Increasingly apoptosis is being recognized as an important factor in determining the ability of the osteoprogenitor lineage to generate a sufficient number of osteoblastic cells to maintain adult bone mass. A variety of hormones and growth factor exert some of their effect by regulating the number of cells that are committed to the apoptotic pathway. Current methods for assessing this important pathway requires biochemical or end point histological assays that cannot reveal the cellular or temporal heterogeneity of the process. We have come to appreciate the power that promoter driven GFP marker genes that allows the bone biologist to recognize stages of osteoblastic differentiation in real time is cell culture and is standard paraffin sections of bone. The R21 proposal is designed to bring this same level of cellular discrimination and ease of assessment t to the process of apoptosis. Because apoptosis is primarily mediated by a proteolytic rather than a transcriptional mechanism, we have designed the marker gone to become activated by cellular caspases. A chimeric GFP - ubiquination protein complex will be constructed in which the two components are linked by a caspase sensitive site such that under basal conditions the marker protein is destroyed. However with activation of caspases, the ubiquination domain is cleaved from GFP and the cell develop a fluorescent signal. A number of other features will be added to the marker gene to stabilize the GFP once generated and to allow the marker gene to be introduced into primary cells with retrovector vehicles. If the construct has the desired biological properties in cultured cell lines and primary cells, then transgenic mice will be generated to characterize its activity in intact bone. Success in production of a functional prototype will lead to creation of marker genes using different colors of GFP and promoter fragments that would be of interest to the broad bone biology community. In addition, the concepts developed in this construct will be of values for any marker gone of a biological process that is based on a proteolytic mechanism. ? ?
