The goals of this project are to 1) determine the primary structure of the novel collagens synthesized by cultured mesangial cells; 2) characterize collagen expression in the mesangium of normal and diseased rate glomeruli and 3) isolate clones of differentiated mesangial cells transformed with oncogene-containing retroviruses. To determine the primary structure of the novel collagens synthesized by rat mesangial cells, cDNA will be synthesized from mesangial cell mRNA, inserted into vectors, amplified and screened. cDNA, encoding novel collagenous polypeptides, will be isolated for use as probes on Norther and Southern blots and for DNA sequencing. Synthetic peptides, based on DNA sequences, will be used to generate polyclonal and monoclonal antibodies. These antibodies and cDNA probes along with those available for known collagens will be used to study collagen expression by immunofluorescence and in situ hybridization, in the mesangium of normal rats and of tats with puromycin induced focal and segmental glomerulosclerosis. Retroviruses containing the oncogenes, SV40 large T antigen or the E1A 12S region, spliced to the SV40 promoter and the neomycin phosphotransferase gene will be used to infect primary cultures of glomeruli. Transformed mesangial cells will be studies for differentiated characteristics. These studies will define collagen expression in an experimental model of glomerulosclerosis and will further define the composition of the mesangial matrix. The information gained will be applicable to the study of human glomerular diseases in which mesangial abnormalities are prominent.
| Karimbux, N Y; Rosenblum, N D; Nishimura, I (1992) Site-specific expression of collagen I and XII mRNAs in the rat periodontal ligament at two developmental stages. J Dent Res 71:1355-62 |
