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

基本信息

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
项目类别：
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&apos 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

项目摘要

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.

我们通过建立合作努力来回应 NOT-AG-18-049“AD/ADRD 合作研究” NIA 建立 MODEL-AD 中心的建模能力显着扩大。创建、严格表征并确保下一代动物模型的快速分发制造这些下一代模型的关键障碍是技术限制。对纳入每个新修改等位基因的人类基因组背景的大小进行限制我们明尼苏达大学 (UMN) 的团队开发了基因替换 (GR) 模型-AD 小鼠品系。这些技术使我们能够定期用高达数百 kb 的完整人类直系同源物替换小鼠基因我们使用这项技术生成了一组匹配的微管相关蛋白 Tau 基因。小鼠替换 (MAPT-GR) 系，我们替换了完整的小鼠 Mapt 基因组编码和调控我们已经证实小鼠具有完整的人类 MAPT 基因组序列（190,081bp）。该 MAPT-GR 等位基因的纯合子在内源水平表达人类 tau，并且所有预期的剪接变体在适当的组织中发现了该模型集的完整功能的人类 MAPT 基因的比例。现在包括两个 wt 对照品系（H1 或 H2 MAPT 单倍型）和越来越多的实验品系与 H1 wt 对照线精确匹配，除了我们专门引入的致病变异外其中第一个单倍型目前正在通过 MODEL-AD 进行进一步表征，现在可用于无限制 AD 研究社区 (JAX) 我们此次合作的具体目标是： 1. 生成。基因替换 (GR) 组小鼠品系，其中涉及 AD 病因学的基因已被我们建议为此合作开发 10 个模型集（>20）。 2. 使用已建立的 MODEL-AD 方法表征 GR 系的匹配组，并不受限制地分发。翻译最相关的等位基因将被纳入当前的等位基因中。 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在完整的人类基因序列中发生的情况这些小鼠品系将包含每个基因的所有潜在治疗人类靶标，范围从完整的。基因组 DNA 序列到它们编码的所有 RNA 转录和蛋白质产物。这些匹配组的序列仅在每行中具体改变的序列上有所不同，任何显着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异，任何被发现能有效纠正这些功能障碍的治疗药物都有望产生直接的效果。对患者的治疗价值。