The use of cloning and expression vectors to study and experimentally manipulate individual genes, independent of their normal resident environment, is a central and vital theme in modern molecular genetic experimentation. Due to a number of unique biological attributes, vaccinia virus (VV) would seem to offer an ideal system for such studies. A number of laboratories have demonstrated the feasibility of this approach by constructing recombinant VV strains which contain and express heterologous viral antigens. Such hybrid vaccine strains may prove useful against a variety of human and animal diseases. Unfortunately, the current methodologies employed to construct recombinant VV are slow, time-consuming, expensive, and do not facilitate genetic engineering of the foreign insert. These drawbacks have thus far retarded the development of VV as a generalized eukaryotic expression vector. The experiments outlined in this proposal have three objectives: 1) To streamline the methods employed to construct chimeric genes and to assay their biological activities; 2) To construct insertion plasmids containing dominant selectable markers which will allow direct, one-step, selection of VV recombinants containing foreign inserts; and 3) To use VV as a research tool with which to study gene systems that are not readily amenable to more conventional approaches - RNA viruses (animal and plant), histocompatibility antigens, and immunoglobulin genes. It is anticipated that such studies will provide insights into a number of basic questions pertaining to viral development and gene regulation as well as developing technologies useful for further development and refinement of VV hybrid vaccine strains.
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