Phenotypic screen in mouse embryonic for TGF-beta signaling mutations

描述（由申请人提供）：小鼠中的多种诱变技术可用于理解基因功能和开发人类疾病的动物模型。然而，小鼠遗传学界缺乏一种广泛适用的方法来进行表型筛选，以确定特定生化或遗传途径中的突变。在小鼠胚胎干细胞（ES）中开发基于细胞的表型筛选可以为这些类型的聚焦突变筛选提供关键方法。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。这些信号系统在胚胎发育和疾病中发挥的不同作用表明了一种复杂的调节模式。这些信号在体内环境中如何调节的性质在很大程度上仍然未知。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，以识别和进一步了解这些途径的调节因子。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。在本提案中，将开发试剂以检验以下假设：小鼠ES细胞中的突变可以基于突变基因产物的改变的活性来鉴定，并且这种改变的活性可以使用下游报告基因来测定。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。

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.