Core B: Antibody and Vectorology

摘要 多囊肾（PKD）保护性治疗发展的主要障碍是我们的 对PKD 1（多囊蛋白-1或PC 1）、PKD 2（多囊蛋白-1或PC 1） PKHD 1（多囊蛋白或PD 1）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实验中的关键细胞通路。 模型，然后寻找这些途径的药物成分，目的是开发治疗 减缓疾病进展的药物。该范例需要经验证的PKD试剂的基本工具包 包括抗体、DNA构建体和细胞系。虽然一些工具在一些中心可用， 在美国，调查人员通常只能有限地接触到他们。PKD研究中存在未满足的需求 社区提供经验证且易于获得的PKD试剂。此外，许多新来的调查人员 该领域可能没有利用这些工具来深入分析PKD蛋白的经验。主要 核心B的目标是通过提供一套全面的经验证的PKD来满足这些需求 沿着试剂和技术援助。这利用了我们已经建立的专业知识， 在过去的二十年里，我们显着扩大了试剂库 最后一次融资。核心B由两个高度集成的组件组成。“抗体” “验证”组件将提供经验证的PC 1和PD 1抗体，并提供以下方面的专家建议： 如何使用试剂此外，我们将开发新的PC 1和PD 1抗体， 使用新策略（折叠结构域抗原）对天然蛋白质具有最高的特异性。的 “病媒学”部分将使调查人员能够立即获得大量的 用于野生型和突变体PKD蛋白和CRISPR PKD基因组编辑的质粒表达载体 载体，并提供定制的PKD表达载体与新开发的标签和/或工程 突变。我们还将提供以一种表达这些蛋白质的同基因MDCK和IMCD细胞系。 稳定和可诱导方式。核心还将协助我们的研究基地利用我们的 试剂具体目的是：目的1：提供一组经验证的PC 1抗体， 检测内源性蛋白质。目标2：提供一组经过验证的PD 1抗体，可以检测 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蛋白生物化学的培训。在 总而言之，核心B的使命是有效消除PKD研究的技术障碍，以便 促进有助于开发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.