Use of Vaccinia Virus as A Cloning and Expression Vector

Hruby, Dennis

Abstract

The use of cloning and expression vectors to study and experimentally manipulate individual genes, independent of their normal resident environment, has become a cental 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 including our own, have demonstrated the feasibility of this approach by constructing recombinant VV strains which contain and express heterologous viral antigens. Such hybrid vaccine strains promise to prove useful in the prevention of a variety of human and animal diseases. Unfortunately, the current methodologies employed to construct recombinant VV are slow, labor intensive, expensive, and do not facilitate genetic engineering of the foreign insert. These drawbacks have thus far retarded the development of VV as generalized eukaryotic cloning and expression vector. The experiments outlined in this proposal have three basic objectives: 1) To extend our ongoing studies on new and novel methods for introducing foreign genes into VV. These approaches include the use of dominant selectable markers and/or bacteriophage vectors which will allow the heterologous inserts to be readily manipulated and then recombined into VV in a direct selectable manner; 2) to examine additional VV promoters and insertion sites for their utility in expressing foreign genes; and most importantly, 3) To continue to use VV as a research tool with which to express and study gene systems that are not readily amenable to conventional approaches such as cellular neuropeptides, neurobiological effectors, and surface markers of bacterial pathogens. It is anticipated that such studies will provide insights into a number of basic questions pertaining to viral development, cellular gene expression and regulation, as well as developing technologies useful for the further development and refinement of VV hybrid vaccine strains.