Cell Model & Assay Core

P30中心的囊性纤维化研究人员需要获得专业知识和专门设备来研究CFTR的表达和功能。对于已建立的CF实验室和CF研究的新实验室，已经确定需要开发表达突变型和野生型CFTR的新型细胞系，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： 具体目标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。这些将包括来自鼠肺的原代细胞和新的慢病毒转导的细胞系。后者包括用于CFTR蛋白质组学和结构研究以及用于高通量筛选以发现下一代CF药物的创新细胞模型。 具体目标2，在上述细胞模型（目标1）中进行CFTR功能测定所需的协助、设备和专业知识将包括生物物理技术（膜片钳）。需要这些测定来测试新策略拯救突变CFTR通道活性和调节的功效以及定义潜在机制。 核心将有助于开发/验证新的CF细胞模型;提供编码特定CFTR突变的原代鼠气道上皮细胞;并协助研究人员进行实验，以测试新操作对突变CFTR的影响（例如，DeltaF 508）蛋白稳定性和通道功能。涉及CFTR翻译后修饰的蛋白质组学研究的合作研究，从CFTR的第一个胞质环中发现特异性阻断NBD 1-TMD 1结合的肽，以及许多其他NIH资助的项目也将得到核心的帮助。 核心A将通过为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表达和功能测定来促进跨学科研究，并有助于CF发病机制和实验治疗的创新研究。

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.