In response to PA-02-013, Pilot and Feasibility Program in Urology, we propose a two-year feasibility study of a hybrid promoter-enhancer with a cell-specific, androgen-regulated expression profile. This work is based on the previous demonstration that a Kap15421I-IAGT transgene (1542 bp of the kidney androgen-regulated protein gene promoter fused to 9/7 kb of the human angiotensinogen gene) was expressed specifically in the proximal convoluted tubule (PCT) epithelial cells of the kidney and retained androgen inducibility similar to the endogenous Kap gene. However, Kap1542 promoter activity was not transferable to other genes in vivo or in vitro, suggesting that DNA elements within the HAGT gene contributed to the pattern of expression of the transgene. We have identified essential elements of this hybrid promoter/enhancer element and propose to test the feasibility of using it as a research tool for PCT cell-specific expression of any heterologous gene. We propose three Specific Aims in which the promoter/enhancer elements are studied further to optimize PCT cell specificity and androgen responsiveness of the regulatory "cassette", these elements are tested in vivo in transgenic mice using a reporter gene, and finally the cassette shown to be active in vitro and in vivo will be used to drive Cre recombinase expression in PCT cells. This study will provide valuable tools for further studies based on the direct expression of genes in the PCT cells or on the genetic cross breeding of the PCT-specific Cre recombinase line with lines of mice containing floxed genes that will result in the targeted deletion (or activation) of genes of interest. Such research tools will facilitate the creation of animal models of renal diseases including renal cell carcinoma through the androgen-regulated, PCT cell-specific expression of SV40 T antigen, and studies of the effects of inactivation of specific genes such as VHL, the gene for Von Hippel Landau disease or the androgen receptor for which systemic inactivation (i.e., in the testicular feminized mouse, Tfm/Y) has effects that mask the role of androgens in renal growth, development and function. The proposed research addresses several of the research goals of the Program Announcement.
NIH ExPorter
NIH ExPorter