Transgenic Mice- Recomb., Fate-Mapping,and Ca2+signaling

描述(由申请人提供)：这项建议的目的是开发、验证和利用新的转基因小鼠模型来研究生殖道器官神经肌肉调节的细胞和分子细节。GU器官复杂的多细胞特性是理解细胞相互作用的重要障碍，细胞相互作用对GU器官生理学、病理生理学和转化至关重要。小鼠的功能获得和功能丧失转基因可能已经成为确定哺乳动物单个基因在体内功能的最具信息量的单一过程，在接下来的20年里，生物学的主要努力之一将是通过选择性地改变小鼠基因组来对哺乳动物基因组进行功能注释。尽管这些方法越来越多地被用来探索细胞和器官功能，而且越来越多的小鼠可以在其中插入特定基因的重组酶识别序列，以便有选择地使这些基因失活，但这些方法中很少有人被设计成针对GU细胞的。这项PAR建议寻求应用转基因和重组蛋白工程的方法，为研究GU系统的细胞提供工具，并利用这些小鼠来检查GU功能的各个方面。将创建不同的转基因系，其中特定的细胞系表达：1)配体激活的Cre重组酶转基因(Cre-ER(T2))，其中Cre重组酶处于细胞特异性和时间调控下，用于条件突变和钙敏感荧光研究；2)双顺反子构建体，由增强型绿色荧光蛋白(EGFP)和Cre重组酶组成，用于条件突变、命运定位和细胞分选，用于单细胞或基因表达研究；以及3)基于GFP的钙敏感荧光蛋白(G-CaMPS1.6x和C-CaMPS1.3)，用于细胞功能和信号的谱系特异性研究。所有的小鼠品系都将被定性，并提供给科学界，以加快条件突变、命运图谱和体内成像技术在胃肠道细胞研究中的使用。我们将利用小鼠来解决与神经肌肉传递以及泌尿生殖道Cajal间质细胞(ICC)的发育和功能相关的重要问题。

DESCRIPTION (provided by applicant): 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.