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

用于 MODEL-AD 的全人类基因替换小鼠系的匹配组

• 批准号: 10468368
• 负责人: MICHAEL D KOOB
• 金额: $ 132.92万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10468368
• 关键词: Alleles; Alzheimer' s disease related dementia; Amyloid beta-Protein; Animal Model; Archives; Brain; Code; Collaborations; Communities; DNA Sequence; Development; Ensure; Etiology; Evaluation; Functional disorder; Gene Cluster; Genes; Genetic Transcription; Genomic DNA; Genomics; Goals; Haplotypes; Homologous Gene; Human; Immunohistochemistry; MAPT gene; Methods; Minnesota; Modeling; Molecular; Molecular Disease; Mus; Mutation; Nucleic Acid Regulatory Sequences; Orthologous Gene; Pathogenicity; Patients; Phenotype; Process; Promoter Regions; Proteins; Public Health; RNA; RNA Splicing; Research; Research Personnel; Risk; Ships; Techniques; Technology; Therapeutic; Therapeutic Agents; Tissues; Universities; Untranslated RNA; Variant; base; cohort; design; effective therapy; frailty; gene product; gene replacement; genetic risk factor; implementation design; mouse model; next generation; risk variant; tau Proteins; therapeutic target; tool; transcriptome

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.

我们正在通过建立合作努力来响应NOT-AG-18-049“AD/ADRD合作研究”， 大大扩展了MODEL-AD中心的建模能力。AD-MODEL由NIA建立， 创建、严格表征并确保下一代晚期乳腺癌动物模型的快速分发 AD发作。制造这些下一代模型的关键障碍是技术限制， 对掺入到每个新的修饰等位基因中的人类基因组背景的大小进行限制， MODEL-AD鼠标线。我们明尼苏达大学（UMN）的研究小组开发了基因替代（GR） 这些技术使我们能够常规地将小鼠基因替换为数百kb的完整人类直系同源物， 在尺寸上。我们使用这种技术来产生一组匹配的微管相关蛋白Tau基因- 在小鼠的MAPT-GR替代系中，我们替换了完整的小鼠Mapt基因组编码和调控基因。 区域（156，547 bp）与完整的人MAPT基因组序列（190，081 bp）。我们已经证实老鼠 该MAPT-GR等位基因纯合以内源水平表达人tau以及所有预期的剪接变体 在适当的组织中发现，并且以全功能人MAPT基因预期的比例存在。该模型集 现在包括两个野生型对照系（H1或H2 MAPT单倍型）和越来越多的实验系， 精确匹配H1野生型对照线，除了我们专门引入的致病性变体， 单倍型。第一条生产线目前正在MODEL-AD进一步表征，现在可用于 AD Research Community Without Restriction（JAX）我们这次合作的具体目标是：1。生成 基因置换（GR）小鼠品系的集合，其中涉及AD病因学的基因已经被 被它们的人类同源物所取代我们建议为这次合作开发10套模型（>20套 共线）。2.使用已建立的MODEL-AD方法表征GR线的匹配集， 不受限制地分发。最相关的等位基因将被纳入电流 MODEL-AD“基本模型”。这些GR系将使我们和其他AD研究人员能够评估分子影响， 致病性突变和风险变异的背景下，他们发生在全人类基因序列中， 患者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，范围从完整的 基因组DNA序列与它们编码的所有RNA转录和蛋白质产物相关。因为基因组 这些匹配组的序列将仅在每一行中特异性改变的序列处不同，任何显著的 这些品系之间的分子差异可以确信地归因于实验品系中的风险变体， 并且任何被发现能有效纠正这些功能障碍的治疗剂都可以预期具有直接的 对患者的治疗价值。