Cell Model & Assay Core

P30中心内的囊性纤维化研究人员需要获得专业知识和专门的设备来研究CFTR的表达和功能。无论是已建立的CFR实验室，还是那些新加入CFTR研究的实验室，都需要开发表达突变和野生型CFTR的新细胞系，以及评估CFTR功能所需的专用设备和生物物理方案。我们特别提出两个具体目标： 具体目的1.表达野生型和突变型CFTR的细胞模型将由Core A开发和提供，其中将包括来自小鼠肺的原代细胞和新型慢病毒转导细胞系。后者包括用于CFTR蛋白质组和结构研究的创新细胞模型，以及用于发现下一代CF药物的高通量筛选。 具体目标2，在上述细胞模型中进行CFTR功能分析所需的援助、设备和专业知识(目标1)将包括生物物理技术(膜片钳)。这些分析是测试新操作拯救突变的CFTR通道活动和调节的有效性以及确定潜在的机制所必需的。 该中心将帮助开发/验证新的CF细胞模型；提供编码特定CFTR突变的原代小鼠呼吸道上皮细胞；并协助研究人员进行实验，以测试新操作对突变CFTR(例如，DeltaF508)蛋白质稳定性和通道功能的影响。合作研究涉及CFTR翻译后修饰的蛋白质组学研究，从CFTR的第一个胞液环路发现特异性阻断NBD1-TMD1结合的多肽，以及许多其他由NIH资助的项目也将得到核心的协助。 CORE A将通过向不太熟悉必要技术的研究人员提供有价值的新细胞模型和CFTR表达和功能分析来促进跨学科研究，并为CF发病机制和实验治疗的创新研究做出贡献。

Cystic fibrosis investigators within the P30 Center require access to expertise and specialized equipment for their studies of CFTR expression and function. For both established CF laboratories and those new to CF research, a need has been identified for developing novel cell lines expressing mutant and wild type CFTR, and for dedicated equipment and biophysical protocols necessary to assess CFTR function. In particular, we propose two specific aims: Specific Aim 1. Cellular models expressing wild type and mutant CFTR will be developed and provided by Core A. These will include primary cells from murine lungs and novel lentiviral-transduced cell lines. The latter include innovative cell models useful for CFTR proteomic and structural studies and for high throughput screens to discover the next generation of CF drugs. Specific Aim 2, Assistance, equipment and expertise necessary to perform functional assays of CFTR in the above cell models (Aim 1) will include biophysical techniques (patch clamp). These assays are required to test the efficacies of new maneuvers to rescue mutant CFTR channel activity and regulation as well as to define the underlying mechanisms. The Core will aid in the development/validation of new CF cell models; provide primary murine airway epithelial cells encoding specific CFTR mutations; and assist investigators with their experiments to test the effects of new maneuvers on mutant CFTR (e.g., DeltaF508) protein stability and channel function. Collaborative studies involving proteomic studies of CFTR post-translational modifications, discovery of peptides from the first cytosolic loop of CFTR that specifically block NBD1-TMD1 binding and numerous other NIH funded projects will also be assisted by the Core. Core A will foster interdisciplinary research by providing valuable new cell models and assays of CFTR expression and function to investigators less familiar with the requisite techniques, and contribute to innovative studies of CF pathogenesis and experimental therapy.