Phenotypic screen in mouse embryonic for TGF-beta signaling mutations

描述(由申请人提供)：在小鼠身上的各种突变技术可用于了解基因功能和开发人类疾病的动物模型。然而，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，以确定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这些类型的聚焦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。这些信号系统在胚胎发育和疾病中扮演的不同角色表明了一种复杂的调控模式。这些信号是如何在体内调节的，其本质在很大程度上仍不清楚。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，以识别和进一步了解这些途径的调节因子。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。在这项提议中，将开发试剂来检验这样的假设，即可以根据突变基因产物的变化的活性来识别小鼠ES细胞中的突变，并且可以使用下游报告来检测这种变化的活性。将开发一种筛选策略来鉴定在小鼠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胚胎干细胞。基于荧光素酶的报告将被用来读出胚胎干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。

DESCRIPTION (provided by applicant): A variety of mutagenesis techniques in the mouse are available to understand gene function and to develop animal models of human disease. However, 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 these types of 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. The diverse roles that these signaling systems play in both embryonic development and disease suggests a complex mode of regulation. The nature of how these signals are regulated in an in vivo context is still largely unknown. A directed mutation screen of TGF-beta-related signaling processes would generate valuable animal models to identify and further understand the regulators 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. In this proposal, reagents will be developed to test the hypothesis that a mutation in mouse ES cells can be identified based on the altered activity of the mutant gene product, and that this altered activity can be assayed using a downstream reporter. 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.