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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。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推进小鼠作为遗传系统的效用。特别是，小鼠遗传学界一直缺乏一种广泛适用的方法来进行表型筛选，以识别特定生化或遗传途径中的突变。基于细胞的小鼠胚胎干（ES）细胞表型筛选的发展可以为聚焦突变筛选提供关键的方法。tgf - β相关信号通路将是胚胎干细胞定向表型筛选的优秀候选者。这些信号通路在包括肿瘤进展在内的多种疾病状态中发挥关键作用。tgf - 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控。许多介导tgf - β信号传导的成分存在于小鼠18号染色体上，这表明对tgf - β信号突变进行染色体定向表型筛选的可能性。方法：将开发一种筛选策略，用于鉴定小鼠18号染色体上tgf - β超家族信号的直接早期反应组分中携带化学诱导突变的胚胎干细胞。基于荧光素酶的报告基因将被用作胚胎干细胞对tgf - β信号反应性的读数。诱变将由乙基亚硝基脲完成，这是一种在胚胎干细胞中非常有效的诱变剂。为了解决隐性突变，cre - loxp介导的有丝分裂重组系统将被用于产生纯合的突变18号染色体细胞。本研究提出的基于细胞的突变筛选策略可以应用于小鼠胚胎干细胞中任何活跃的生化途径，以产生用于体内分析的小鼠系。

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.