Matched sets of full human gene-replacement mouse lines for MODEL-AD

我们正在响应NOT-AG-18-049《AD/ADRD的协作研究》，通过建立一个协作努力来 大幅扩展模型的建模能力--AD中心。模型-AD是由NIA建立的，以 创建、严格描述并确保最近快速分发下一代动物模型 公元一开始。在制造这些下一代模型方面取得进展的关键障碍是技术限制， 对合并到每个新修改的等位基因中的人类基因组上下文的大小进行限制 模型-AD小鼠品系。我们明尼苏达大学(UMN)的团队已经开发出基因替换(GR)。 允许我们常规地用长达几百kb的完整人类同源基因替换老鼠基因的技术 在尺寸上。我们利用这项技术产生了一组匹配的微管相关蛋白Tau基因- 替换小鼠(MAPT-GR)系，在其中我们替换了完整的小鼠MAPT基因组编码和调控 人MAPT全基因组序列(190,081bp)。我们已经证实老鼠 这种MAPT-GR等位基因纯合在内源性水平表达人类tau，所有预期的剪接变体 在适当的组织中发现，并且在完全功能的人类MAPT基因的预期比例中被发现。这套模型 现在包括两个wt控制系(H1或H2 MAPT单倍型)和越来越多的实验品系 精确匹配h1重量控制线，除了我们特别引入的致病变异体 单倍型。这些生产线中的第一条目前正在由型号-AD进一步表征，现在可以用于 AD研究社区无限制(JAX)。我们这次合作的具体目标是：1.产生 与阿尔茨海默病病因学相关的基因替换(GR)组小鼠 取而代之的是它们的全人类同源基因。我们建议为此协作开发10个模型集(&gt；20 总行数)。2.使用所建立的模型-AD方法和 不受限制地分发。最具翻译相关性的等位基因将被合并到当前的 模型-AD“基本模型”。这些GR线将使我们和其他AD研究人员能够评估分子影响 在发生致病突变和风险变异的整个人类基因序列的背景下 病人。这些小鼠品系将包含每个基因的所有潜在的人类治疗靶点，从完整的 基因组DNA序列到所有的RNA转录和他们编码的蛋白质产品。因为基因组 这些匹配组的序列将仅在每一行中特别改变的序列上不同，任何显著的 这些品系之间的分子差异可以肯定地归因于实验品系中的风险变量， 任何被发现能有效纠正这些功能障碍的治疗剂都有望直接 对患者的治疗价值。

We are responding to NOT-AG-18-049 “Collaborative Studies on AD/ADRD” by establishing a collaborative effort to significantly expand the modeling capacity of the MODEL-AD Center. MODEL-AD was established by the NIA to create, rigorously characterize and ensure the rapid distribution of the next generation of animal models of Late Onset AD. Critical barriers to progress in making these next generation models are technique limitations that have put restrictions on the size of the human genomic context incorporated into each of the new modified alleles in the MODEL-AD mouse lines. Our group at the University of Minnesota (UMN) has developed Gene Replacement (GR) technologies that allow us to routinely replace mouse genes with their full human orthologs up to several hundred kb in size. We used this technology to generate a matched set of Microtubule Associated Protein Tau Gene- Replacement (MAPT-GR) lines of mice in which we replaced the full mouse Mapt genomic coding and regulatory region (156,547bp) with full human MAPT genomic sequences (190,081bp). We have confirmed that mice homozygous for this MAPT-GR allele express human tau at endogenous levels, and that all expected splice variants are found in the appropriate tissues and in ratios expected for the fully functional human MAPT gene. This model set now includes two wt control lines (H1 or H2 MAPT haplotype) and a growing number of experimental lines that precisely match the H1 wt control line except for the pathogenic variant that we specifically introduce into that haplotype. The first of these lines are currently being further characterized by MODEL-AD and are now available to the AD research community without restriction (JAX). Our specific aims for this collaboration are to: 1. Generate Gene-Replacement (GR) sets of mouse lines in which genes involved in the etiology of AD have been replaced by their full human homologs. We are proposing to develop 10 model sets for this collaboration (>20 total lines). 2. Characterize matched sets of GR lines using the established MODEL-AD methods and distribute without restriction. The most translationally relevant alleles will be incorporated into the current MODEL-AD “base model”. These GR lines will allow us and other AD researchers to evaluate the molecular impact of pathogenic mutations and risk variants within the context of the full human gene sequence in which they occur in patients. These mouse lines will contain all potential human therapeutic targets for each gene, ranging from the full genomic DNA sequences to all RNA transcription and protein products that they encode. Because the genomic sequences of these matched sets will differ only at sequences specifically changed in each line, any significant molecular differences between these lines can confidently be attributed to the risk variant in the experimental lines, and any therapeutic agents found to effectively correct these dysfunctions could be expected to have direct therapeutic value to patients.