PHENOTYPIC SCREENS OF THE TGF-BETA TUMOR SUPPRESSOR PATHWAY IN MOUSE ES CELLS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。前言：小鼠是了解体内基因功能和建立人类疾病动物模型的关键遗传系统。需要新的方法来进一步提高小鼠作为遗传系统的效用。特别是，小鼠遗传学社区一直缺乏一种广泛适用的手段来进行表型筛选，以识别特定生化或遗传途径中的突变。小鼠胚胎干细胞基于细胞的表型筛选的发展可以为集中突变筛选提供关键的方法学。转化生长因子-β相关的信号通路将是ES细胞定向表型筛选的极佳候选者。这些信号通路在包括肿瘤进展在内的多种疾病状态中起着关键作用。转化生长因子-β相关信号过程的定向突变筛选将产生有价值的动物模型，以进一步了解这些途径的调节。许多介导转化生长因子-β信号的成分存在于小鼠的18号染色体上，这表明有可能对转化生长因子-β信号突变进行染色体导向的表型筛选。方法：将建立一种筛选策略，用于鉴定在小鼠18号染色体上的转化生长因子-β超家族信号的即时早期反应成分中携带化学诱导突变的ES细胞。基于荧光素酶的报告将被用来读出ES细胞对转化生长因子-β信号的反应性。诱变将用乙基亚硝脲完成，乙基亚硝脲是ES细胞中的一种高效诱变剂。为了解决隐性突变，将利用Cre-loxP介导的有丝分裂重组系统来产生对诱变的18号染色体纯合的细胞。本建议发展的基于细胞的突变筛选策略可以应用于小鼠ES细胞中任何活跃的生化途径，以产生用于体内分析的小鼠品系。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Introduction: The mouse is a critical genetic system to understand in vivo gene function and to develop animal models of human disease. New methods will be needed to further advance the utility of the mouse as a genetic system. In particular, the mouse genetics community has lacked a broadly applicable means of performing phenotypic screens to identify mutations in specific biochemical or genetic pathways. The development of cell-based phenotypic screens in mouse embryonic stem (ES) cells could provide the critical methodology for focused mutation screens. The TGF-beta-related signaling pathways would be excellent candidates for directed phenotypic screens in ES cells. These signaling pathways play a critical role in a variety of disease states, including tumor progression. A directed mutation screen of TGF-beta-related signaling processes would generate valuable animal models to further understand the regulation of these pathways. Many of the components that mediate TGF-beta signaling are present on mouse chromosome 18, suggesting the possibility of a chromosomal-directed phenotypic screen for TGF-beta signaling mutations. Methods: A screening strategy will be developed for identifying ES cells that carry chemically induced mutations in the immediate early response components of TGF-beta superfamily signaling on mouse chromosome 18. A luciferase based reporter will be used as a readout of the responsiveness of ES cells to TGF-beta signaling. Mutagenesis will be accomplished with ethylnitrosourea, a highly effective mutagen in ES cells. To resolve recessive mutations, a Cre-loxP-mediated mitotic recombination system will be utilized to generate cells that are homozygous for a mutagenized chromosome 18. Cell-based mutation screening strategies as developed in this proposal can be applied to any active biochemical pathway in mouse ES cells, to generate mouse lines for analysis in vivo.