The aim of this proposal is to develop, validate, and utilize new transgenic mouse models for the study of the cellular and molecular details of neuromuscular regulation of organs of the genitourinary tract. The complex, multicellular nature of GU organs is a significant barrier to the understanding of the cellular interactions that are critical to GU organ physiology, pathophysiology, and transformation. Gain-of-function and loss-of-function transgenesis in the mouse has emerged as perhaps the single most informative process in determining the in vivo function of individual genes in mammals, and over the next two decades one of the major efforts of biology will be the functional annotation of the mammalian genome through selective alterations of the mouse genome. Despite the increasing use of these methods to probe cellular and organ function, and the increasing availability of mice in which recombinase recognition sequences have been inserted for specific genes so that the genes can be selectively inactivated, few of these approaches have been designed to target cells of the GU. This PAR proposal seeks to apply methods of transgenesis and recombinant protein engineering to provide tools for the study of cells of the GU system, and to use these mice to examine aspects of GU function. Separate transgenic lines will be created in which specific cellular lineages express: 1) a ligand -activated Cre recombinase transgene (Cre-ER (T2)) in which Cre recombinase is under cell specific and temporal control for conditional mutagenesis and Ca2+ -sensitive fluorescence studies; 2) a bicistronic construct consisting of enhanced Green Fluorescent Protein (eGFP) and Cre recombinase for conditional mutagenesis, fate mapping, and cell sorting for single cell or gene expression studies; and 3) GFP - based Ca2+ -sensitive fluorescent proteins (G-CaMPS1.6x and C-CaMPS1.3) for lineage specific studies of cell function and signaling. All lines of mice will be characterized and made available to the scientific community to accelerate the use of conditional mutagenesis, fate mapping, and in-vivo imaging techniques in the study of cells of the GU tract. We will use the mice to address important questions relating to neuromuscular transmission and the development and function of Interstitial Cells of Cajal (ICC) in the urogenital tract.
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