Platelet-Derived Growth Factor (PDGF) is encoded by two genes, A and B. The A and B gene products dimerize to form any of three PDGF isoforms (A:A, A:B or B:B) in which the two subunits are linked through disulfide bonds. We constructed mutations within cDNA clones of PDGF A. The mutated PDGF A proteins dimerize with wild type PDGF subunits (either A or B) but the resulting heterodimers are inactive. Thus, our mutants encode trans dominant suppressors of PDGF gene expression. The research described here is framed around this preliminary work and it has three objectives. The first objective is to create PDGF deficient mice. These mice may display the in vivo functions of PDGF (poorly understood right now) by default. The construction of PDGF-deficient mice will involve no more than standard transgenic mouse technology. To begin, we will make a mouse which does not contain PDGF in its platelets. A tissue-specific promoter for platelet factor 4 will be used to drive PDGF suppressor gene expression within bone marrow megakaryocytes - the platelet progenitor cells. The second objective is to create specific, soluble, inhibitors of PDGF. The translation products of PDGF dominant suppressor genes will be used as a point of departure. Using insect virus expression vectors, we will produce and isolate heterodimers of one """"""""wild type"""""""" PDGF subunit covalently linked to one """"""""suppressor"""""""" subunit. According to current understanding of the PDGF:receptor interaction, these heterodimeric proteins should inhibit the binding of wild type PDGF to its receptor. These suppressor proteins, or derivatives of them, could one day be used to treat hyperproliferative disorders of connective tissue such as atherosclerosis, arthritis, and glioma. The third objective sounds derivative but it's really not. Using structural lesions similar to those we created within the PDGF cDNAs, we will develop dominant suppressors of TGF-beta and go on to construct TGF-beta deficient mice.There are two good reasons to do this. The first is that by mating the appropriate TGF-beta deficient mouse to a PDGF deficient mouse, we can test a very specific hypothesis-that the function of PDGF and TGF-beta within platelet cells in vivo is to promote the process of wound healing. The second reason is that TGF-beta actually inhibits the growth of most cell types. With TGF-beta deficient mouse strains, we could test the hypothesis that growth inhibitory factors, such as TGF-beta, function as anti-oncogenes.
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