The goal of this grant is to develop two new techniques; one is for the spatial and temporal control of transgene expression in mice; and the other for generation of transgenic mice. There are several difficulties with introducing, and expressing a transgene in mice. These include unpredictable insertion site(s), unpredictable copy number, and unpredictable expression. The first technique to be developed will use a bacteriophage RNA polymerase (RNAP) to regulate transgene expression in a temporal and spatial manner. The RNAP is fused to an estrogen or progesterone ligand-binding domain such that it will be localized to the cytoplasm in the absence of ligand but translocate to the nucleus when ligand is added. Thus, a cDNA under transcriptional control of the RNAP promoter can only be expressed when the RNAP localizes to the nucleus. Temporal control is achieved by application of the ligand and spatial control is achieved by placing the hybrid RNAP/ligand binding domain protein under control of a tissue-specific promoter. The second technique uses modified gene-targeting approaches to allow the insertion of a cDNA into a predetermined gene such that the endogenous promoter will control cDNA expression. Described will be the selection methods needed to generate the engineered embryonic stem cells to generate the chimeric mice. The skin is an ideal organ in which to develop these techniques. First, there are a number of well- characterized cutaneous specific promoters available. Secondly, the skin is accessible, permitting easy tissue sampling without euthanasia or surgery and also visual inspection for phenotypic changes. Further, the skin is often considered as a site for the introduction of genes, such as clotting factors, in gene-therapy procedures. Thus these procedures, if rendered into practice, could serve as a quick and relatively easy method to determine proof-of-principle for gene therapy applications.
