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

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 小鼠是了解体内基因功能和开发人类疾病动物模型的关键遗传系统。 将需要新的方法来进一步推进小鼠作为遗传系统的效用。 特别是，小鼠遗传学界缺乏一种广泛适用的方法来进行表型筛选，以确定特定生物化学或遗传途径中的突变。 基于细胞的小鼠胚胎干细胞表型筛选技术的发展为基因突变筛选提供了重要的方法学基础。 TGF-β相关的信号通路将是ES细胞中定向表型筛选的极好候选者。 这些信号通路在各种疾病状态中发挥关键作用，包括肿瘤进展。TGF-β相关信号传导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控。 许多介导TGF-β信号传导的组分存在于小鼠18号染色体上，这表明可能存在TGF-β信号传导突变的染色体定向表型筛选。 研究方法：将开发一种筛选策略，用于鉴定在小鼠18号染色体上TGF-β超家族信号传导的立即早期反应组分中携带化学诱导突变的ES细胞。 基于荧光素酶的报告基因将用作ES细胞对TGF-β信号传导的响应性的读出。 将使用乙基亚硝基脲（ES细胞中的高效诱变剂）完成诱变。 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于诱变的染色体18纯合的细胞。 本提案中开发的基于细胞的突变筛选策略可应用于小鼠ES细胞中的任何活性生化途径，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.