Core B: Antibody and Vectorology

摘要 多囊肾病(PKD)保护性疗法发展的主要障碍是我们的 对PKD1(多囊蛋白-1或PC1)、PKD2改变的细胞通路的了解有限 (多囊蛋白-2或PC2)或PKHD1(多聚蛋白或PD1)基因(统称为PKD基因/蛋白质)是 变异了。总的来说，研究人员已经确定了细胞培养系统和动物体内的关键细胞通路。 模型，然后寻找这些通路的可用药成分，目标是开发治疗 可以减缓疾病进展的药物。这一范例需要一个经过验证的PKD试剂的基本工具包 这包括抗体、DNA结构和细胞系。尽管一些工具在以下几个中心可用 在美国，调查人员接触这些文件的机会往往有限。在PKD研究中有一个未得到满足的需求 提供经过验证的、现成的PKD试剂社区。此外，许多新手调查人员 该领域可能没有利用这些工具深入分析PKD蛋白质的经验。主 核心B的目标是通过提供一套全面的经过验证的PKD来满足这些需求 试剂和技术援助。这利用了我们已经建立的专业知识 在过去的二十年里，我们在过去的二十年里显著扩大了试剂仓库 最后一个资助期。核心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，以及专家对 如何使用试剂。此外，我们将开发新的PC1和PD1抗体，它们具有 使用一种新的策略(折叠结构域抗原)，对天然蛋白具有最高的特异性。这个 “向量学”组件将允许调查人员立即访问广泛的 野生型和突变型PKD蛋白的表达载体及CRISPR PKD基因组编辑 载体，并提供带有新开发的标签和/或工程的自定义PKD表达载体 突变。我们还将提供表达这些蛋白的等基因MDCK和IMCD细胞系 稳定、诱人的时尚。核心还将帮助我们的研究基地利用我们的 试剂。具体目标是：目标1：提供一组有效的PC1抗体，可以 检测内源性蛋白质。目的2：提供一组有效的PD1抗体，可以检测到 内源性蛋白质。目的3：提供PKD表达载体和CRISPR基因组编辑 向量。目的4：建立稳定、可诱导表达PKD蛋白的肾细胞模型。 和PKD基因敲除。目的5：提供PKD蛋白质生物化学培训工作坊。在……里面 总结，核心B的使命是有效消除PKD研究的技术障碍，以便 促进有助于发展PKD保护性疗法的发现。

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.