Core B: Antibody and Vectorology

核心 B：抗体和载体学

• 批准号: 9149230
• 负责人: Feng Qian
• 金额: $ 16.51万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9149230
• 关键词: Animal Model; Antibodies; Antigens; Baltimore; CRISPR/Cas technology; Cell Culture System; Cell Line; Cell model; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Communities; Complex; Coupled; Custom; DNA; Development; Disease Progression; Educational workshop; Engineering; Funding; Gene Proteins; Genes; Goals; Lyase; Mission; Mutate; Mutation; N-terminal; Oryctolagus cuniculus; PKD2 protein; Pathway interactions; Pectins; Plasmids; Polycystic Kidney Diseases; Proprotein Convertase 1; Proprotein Convertase 2; Protein Biochemistry; Proteins; Protocols documentation; Publications; Rattus; Reagent; Research; Research Personnel; S-nitro-N-acetylpenicillamine; Services; Specificity; Therapeutic Agents; Training; Validation; abstracting; base; experience; expression vector; extracellular; genome editing; inducible gene expression; kidney cell; knockout gene; meetings; mutant; novel; novel strategies; polycystic kidney disease 1 protein; repository; response; success; tool; vector

Abstract A major obstacle to the development of protective therapies for polycystic kidney disease (PKD) has been our limited understanding of the cellular pathways that are altered when the PKD1 (polycystin-1 or PC1), PKD2 (polycystin-2 or PC2) or PKHD1 (polyductin or PD1) genes (collectively referred to as PKD genes/proteins) are mutated. In general, researchers have identified critical cellular pathways in cell culture systems and in animal models, and then sought druggable components of these pathways with the goal of developing therapeutic agents that would slow disease progression. This paradigm requires a basic tool kit of validated PKD reagents that includes antibodies, DNA constructs and cell lines. Although some tools are available at a few centers in the US, investigators frequently have limited access to them. There is an unmet need in the PKD Research Community for validated and readily available PKD reagents. In addition, many investigators who are new to the field may not have the experience to leverage these tools for in-depth analysis of PKD proteins. The main objective of Core B is to meet these needs by providing a comprehensive set of validated PKD reagents along with technical assistance. This takes advantage of expertise that we have established over the past two decades and a reagent repository that we have significantly expanded during the last funding period. Core B is comprised of two highly integrated components. The “antibody validation” component will provide validated PC1 and PD1 antibodies coupled with expert advice on how to use the reagents. Furthermore, we will develop novel PC1 and PD1 antibodies that have the highest specificity against native protein using a novel strategy (folded domain antigens). The “vectorology” component will allow investigators immediate access to an extensive collection of plasmid expression vectors for wild type and mutant PKD proteins and CRISPR PKD genome-editing vectors, and offer custom PKD expression vectors with newly developed tags and/or engineered mutations. We will also provide isogenic MDCK and IMCD cell lines that express these proteins in a stable and inducible fashion. The Core will also assist our research base with utilization of our reagents. The Specific Aims are: Aim 1: To provide a panel of validated PC1 antibodies that can detect endogenous proteins. Aim 2: To provide a panel of validated PD1 antibodies that can detect endogenous proteins. Aim 3: To provide PKD expression vectors and CRISPR genome-editing vectors. Aim 4: To provide renal cell models with stable and inducible expression of PKD proteins, and with PKD gene knockouts. Aim 5: To provide a training workshop in PKD protein biochemistry. In summary, the mission of Core B is to effectively remove technical barriers to PKD research in order to promote discoveries that will facilitate the development of protective therapies for PKD.

抽象的 多囊性肾脏疾病（PKD）开发受保护疗法的主要障碍是我们的 对PKD1（Polycystin-1或PC1），PKD2改变的细胞途径的了解有限 （Polycystin-2或PC2）或PKHD1（polductin或pd1）基因（统称称为PKD基因/蛋白质）是 突变。通常，研究人员已经确定了细胞培养系统和动物中的关键细胞途径 模型，然后闻到这些途径的可吸毒成分，目的是开发治疗 会减慢疾病进展的药物。此范式需要一个基本的工具套件，包括经过验证的PKD试剂 其中包括抗体，DNA构建体和细胞系。尽管在几个中心可以使用一些工具 美国调查人员的访问量经常有限。 PKD研究有未满足的需求 社区，用于经过验证且易于使用的PKD试剂。此外，许多新的调查员是 该领域可能没有经验来利用这些工具来深入分析PKD蛋白。主 核心B的目的是通过提供一组经过验证的PKD来满足这些需求 试剂以及技术帮助。这利用了我们已经建立的专业知识 在过去的二十年中 最后的资金期。 Core B由两个高度集成的组件组成。 “抗体 验证”组件将提供经过验证的PC1和PD1抗体以及有关专家建议 如何使用试剂。此外，我们将开发具有具有的新型PC1和PD1抗体 使用新型策略（折叠域抗原）对天然蛋白质的最高特异性。这 “矢量学”组件将允许调查人员立即访问大量的集合 用于野生型和突变体PKD蛋白和CRISPPR PKD基因组编辑的质粒表达向量 向量，并提供带有新开发标签和/或工程的自定义PKD表达式向量 突变。我们还将提供同基因MDCK和IMCD细胞系，以在A中表达这些蛋白质 稳定且诱导的时尚。核心还将帮助我们的研究基础利用我们 试剂。具体目的是：目标1：提供一组经过验证的PC1抗体 检测内源性蛋白。目标2：提供一组可以检测到的验证的PD1抗体 内源性蛋白质。目标3：提供PKD表达载体和CRISPR基因组编辑 向量。 AIM 4：提供肾细胞模型具有PKD蛋白的稳定且可诱导的表达， 以及PKD基因淘汰赛。目标5：提供PKD蛋白生物化学的培训研讨会。在 总结，核心B的任务是有效消除PKD研究的技术障碍，以便 促进将有助于开发PKD的受保护疗法的发现。