Functional Dissection of Signaling Pathways

描述（由申请人提供）：细胞多样性和对环境变化的反应主要由针对大多数基因转录调控水平的信号网络触发。后基因组时代生物医学研究的一个主要挑战是识别和理解基因调控序列在正常和疾病影响途径中协调基因转录的机制。该计划的目标是开发和商业化的一套验证慢病毒转录报告载体和报告细胞系的最关键的疾病相关的信号转导途径。这些通路特异性报告载体将使研究人员能够研究通路机制，并确定参与人类疾病发病机制的机制变化。在I期资助下，开发了一组慢病毒转录报告载体、用于构建转录因子特异性构建体的技术和高复杂性转录报告文库，并针对HeLa细胞模型中的p53途径进行了验证。开发了基于条形码转录报告子文库和Affyphase GeneChip（TM）微阵列的组合的用于鉴定功能性报告子构建体的高通量技术。一组用于15种不同转录因子的单色转录报告载体和构建体作为商业产品发布。在第二阶段的资助下，我们建议将该计划扩展到开发和商业化一套全面的经验证的慢病毒报告基因构建体，用于25种具有双色荧光素酶和荧光报告基因的充分表征的转录因子，涵盖与各种人类疾病相关的约30种主要信号转导途径。将通过针对由不同治疗剂和信号转导剂处理的不同细胞系阵列筛选高复杂性转录报告子文库以及通过siRNA构建体敲低特定转录因子来进行单个构建体的功能验证。该技术将与克利夫兰临床基金会合作，用于确定药物开发的靶点，旨在恢复一组人类癌细胞系模型中受抑制的p53通路。拟议研究的预期成果将是一套市售的试剂盒，包括慢病毒报告构建体，用于药物筛选应用的稳定转录报告细胞系的集合，不同转录反应元件和转录模块的功能活性数据库，以及几种经验证的抗癌药物靶标。此外，我们还将推出人类和小鼠全基因组转录报告基因文库，这将是发现与疾病状态相关的新型转录因子的有力研究工具。

DESCRIPTION (provided by applicant): The cellular diversity and response to environmental changes are primarily triggered by a signaling network that is directed at the level of transcriptional regulation of most genes. A major challenge for biomedical research in the post-genomic era will be to identify and understand the mechanisms by which gene regulatory sequences coordinate gene transcription in normal and disease-affected pathways. The goal of the proposed program is to develop and make commercially available a set of validated lentiviral transcriptional reporter vectors and reporter cell lines for the most critical disease-related signal transduction pathways. These pathway-specific reporter vectors will allow researchers to study pathway mechanisms and identify changes in the mechanisms involved in the pathogenesis of human diseases. Under Phase I funding, a set of lentiviral transcriptional reporter vectors, technology for construction of transcriptional factor- specific constructs, and high complexity transcriptional reporter libraries were developed and validated for the p53 pathway in the HeLa cell model. High-throughput technology for identification of functional reporter constructs based on combination of bar-coded transcriptional reporter libraries and Affymetrix GeneChip(tm) microarrays was developed. A set of single color transcriptional reporter vectors and constructs for 15 different transcriptional factors was released as commercial products. Under Phase II funding, we propose to extend the program towards the development and commercialization of a comprehensive set of validated lentiviral reporter constructs for 25 well-characterized transcriptional factors with dual color luciferase and fluorescent reporters, covering about 30 major signal transduction pathways associated with a wide range of human diseases. The functional validation of individual constructs will be performed by screening the high complexity transcriptional reporter library against an array of different cell lines treated by different therapeutic and signal transducing agents and by knockdown of specific transcriptional factors by siRNA constructs. The established technology will be applied, in collaboration with the Cleveland Clinic Foundation, for identification of targets for drug development aimed at the restoration of suppressed p53 pathway in a set of human carcinoma cell line models. The anticipated outcomes of the proposed research and will be a commercially available set of kits comprising of lentiviral reporter constructs, a collection of stable transcriptional reporter cell lines for drug screening applications, a database of functional activity of different transcriptional response elements and transcriptional modules, and several validated anticancer drug targets. In addition, we will launch human and mouse genome-wide transcriptional reporter libraries, which will be a powerful research tool for the discovery of novel transcriptional factors associated with disease state.