Cell-specific Gene Targeting in Urologic Tissues

描述(由申请人提供)：为了响应PA-02-013，泌尿学试点和可行性计划，我们提议进行一项为期两年的具有细胞特异性、雄激素调节表达谱的混合启动子-增强子的可行性研究。这项工作是基于先前证明Kap15421I-IAGT转基因(肾脏雄激素调节蛋白基因启动子1542bp与人血管紧张素原基因9/7kb融合)在肾脏近曲小管(PCT)上皮细胞中特异表达，并保持与内源性Kap基因相似的雄激素诱导能力。然而，Kap1542启动子的活性在体内或体外不能转移到其他基因，这表明Hagt基因中的DNA元件有助于转基因的表达模式。我们已经确定了这种混合启动子/增强子元件的基本成分，并建议测试将其用作任何异源基因的PCT细胞特异性表达的研究工具的可行性。我们提出了三个具体目标，进一步研究启动子/增强子元件，以优化PCT细胞特异性和调节“盒”的雄激素反应，这些元件在转基因小鼠体内使用报告基因进行测试，最后，体外和体内活性的盒将用于驱动Cre重组酶在PCT细胞中的表达。本研究将为基于基因在PCT细胞中的直接表达或PCT特异性Cre重组酶品系与含有目的基因的小鼠品系的遗传杂交提供有价值的工具，这些品系将导致目的基因的定向缺失(或激活)。这些研究工具将有助于通过雄激素调节的PCT细胞特异性表达SV40T抗原来建立包括肾细胞癌在内的肾脏疾病的动物模型，以及研究特定基因的失活的影响，如VHL、Von Hippel Landau病的基因或系统失活的雄激素受体(即在睾丸女性化的小鼠中，TFM/Y)具有掩盖雄激素在肾脏生长、发育和功能中的作用的效果。这项拟议的研究涉及该计划公告的几个研究目标。

DESCRIPTION (provided by applicant): 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.