The training plan for the candidate will focus on two phases. Phase I will involve approximately five or six formal courses taking about 45 percent of the candidate's time during the first two years of the program. In addition, during this same time period, the candidate will be spending approximately 45 percent of his time on research-related activities and about 10 percent on clinical responsibilities. His progress will be monitored by the sponsor, as well as a faculty advisory committee which will serve as the Advisory Committee for the annual review, and the evaluation of the candidate's formal research plan which will be developed during the first two years of his training. The transition from phase I to phase II will be controlled by this Faculty Advisory Committee. The research outlined in this application centers on the idea that there has been no attempt to stringently regulate the expression of a transferred gene in an animal. The candidate believes that the enzymes important for lactose metabolism (lac) or lac operon system, a gene repressor/operator system of Escherichia coli (E. coli) could be of value in this regard. It has been established that the lac repressor and operator are functional in mammalian cells in tissue culture, including murine C2C12 myoblasts, and that the inducing agent, isopropyl beta-D- thiogalactoside (IPTG), can reverse repression.Further, the lac system can achieve stringent repression of an operator-linked gene that is stably integrated into a mammalian chromosome, and at least several hundred-fold induction of expression can be achieved with IPTG. Induction is fully reversible with removal of IPTG, and IPTG has been shown to be nontoxic in mice and to be biologically active when administered intraperitoneally. The project proposes to introduce the E. coli lac regulatory system linked to the human growth hormone (hGH) gene into murine C2C12 myoblasts, and implant these engineered cells into mice. The hypothesis is that systemic expression of hGH could then be regulated by intraperitoneal administration of varying amounts of the inducing agent, IPTG. Future work will be to apply gene transfer treatment to humans with inherited protein hormone deficiency states such as idiopathic hGH deficiency.
