Developing Regeneration Resources for a Model Amphibian

为模型两栖动物开发再生资源

• 批准号: 9272459
• 负责人: Jon Scott Thorson
• 金额: $ 57.33万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9272459
• 关键词: Accelerometer; Amaze; Ambystoma; Ambystoma mexicanum; Amphibia; Amputation; Animal Model; Biological; Biological Assay; Biological Process; Biology; CRISPR/Cas technology; Candidate Disease Gene; Cell Lineage; Cell Proliferation; Cells; Chemical Models; Chemicals; Chimeric Proteins; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Competence; Couples; Data; Development; Embryo; Event; FDA approved; Future; Gene Expression; Gene Targeting; Gene Transfer Techniques; Genes; Genetic; Genetic Models; Genetic Screening; Genetic Transcription; Guide RNA; Histology; Hormone use; Injectable; Insertional Mutagenesis; Investigation; Knock-out; Libraries; Mediating; Methods; Modeling; Molecular; Molecular Profiling; Monitor; Mosaicism; Muscle satellite cell; Mutagenesis; Mutation; Natural Products; Natural regeneration; Neuroglia; Organism; Pathway interactions; Pharmacology; Phenotype; Population; Process; Production; Reagent; Recruitment Activity; Regenerative Medicine; Regenerative response; Reporter; Reporting; Research; Research Infrastructure; Resources; Salamander; Signal Pathway; Signal Transduction; Site; Somatic Cell; Stem Cell Research; Stem cells; Tail; Testing; Thyroid Hormones; Time; Tissues; Transforming Growth Factor beta; Transgenic Organisms; Tretinoin; Vertebrate Biology; blastema; chemical genetics; comparative efficacy; design; dosage; feeding; in vivo; inhibitor/antagonist; mutant; nerve stem cell; network models; notch protein; novel; progenitor; promoter; public health relevance; regenerative; screening; stem cell biology; tool; web site; wound; wound epidermis

 DESCRIPTION (provided by applicant): This Multi-PD/PI application seeks to develop resources that will better enable studies of a highly regenerative amphibian, the Mexican axolotl (Ambystoma mexicanum). There is need to probe axolotl regeneration more deeply, with the same powerful approaches that have proven so effective in genetic model organisms. Specific Aim 1 will accomplish the first chemical genetic screen of axolotl regeneration using an embryo tail regeneration assay. Using pre-feeding axolotl embryos that are efficiently reared in micro-titer plates, up to 10,000 soluble chemicals from commercial, clinical-stage, and novel natural products libraries will be tested for impact on tail regeneration. Preliminary data show that the chemical screen and tail regeneration assay are likely to identify new molecules that impact regeneration. Positive hits from this screen, including previously identified inhibitors of Wnt, Tgfβ, and Fgf signaling, will be reported to the community and investigated further under Specific Aim 2, using cellular and transcriptional approaches. In particular, assays will be developed to assess chemical effects on formation of the wound epidermis, which acts as an early signaling center in the recruitment of progenitor/stem cells. Also, assays will be developed to assess cellular proliferation and de- differentiation, two processes that are associated with endogenous regeneration. Genes found to be expressed differently between control and chemically treated embryos will be prioritized for knock out using the CRISPR/Cas9 method. For each gene target, two gRNA pairs will be designed and injected into single cell axolotl embryos. Embryos will be reared to assess viability, and then administered tail amputations to confirm CRISPR gene editing and test for regeneration competence. Embryos associated with CRISPRs that block or cause abnormal regeneration, will be prioritized for founding stable lines. Specific Aim 3 will generate fluorescent reporter lines to assay signaling activity through major pathways known to function in regeneration and will also compare efficacies of two methods for developing gene-specific reporter lines, using as tester loci genes already known to mark cell populations critical for regenerative responses (neural stem cells, glia, muscle satellite cells). The chemical and genetic hits, and biological information arising from this model will be shared through a community website (Sal-Site). The proposed transgenics will be distributed by the Ambystoma Genetic Stock Center. Overall, this project integrates expertise across chemical screening, pharmacology, histology, transcription, transgenesis, and vertebrate biology to discover reagents and develop tools that are needed to enhance the axolotl for stem cell and regenerative biology.

 描述（由申请人提供）：本多PD/PI申请旨在开发资源，以便更好地研究高度再生的两栖动物墨西哥蝾螈（Ambystoma mexicanum）。有必要更深入地探索美西螈的再生，使用在遗传模式生物中证明如此有效的同样强大的方法。具体目标1将使用胚胎尾部再生测定完成蝾螈再生的第一次化学遗传筛选。使用在微量滴定板中有效饲养的预饲养蝾螈胚胎，将测试来自商业，临床阶段和新型天然产物库的多达10，000种可溶性化学物质对尾部再生的影响。初步数据显示，化学筛选和尾部再生试验可能会识别影响再生的新分子。来自该筛选的阳性结果，包括先前鉴定的Wnt、TGFβ和FGF信号传导抑制剂，将报告给社区，并在特定目标2下使用细胞和转录方法进行进一步研究。特别是，将开发测定法以评估对伤口表皮形成的化学作用，伤口表皮在祖细胞/干细胞的募集中充当早期信号传导中心。此外，将开发测定法以评估细胞增殖和去分化，这两个过程与内源性再生相关。在对照和化学处理的胚胎之间发现表达不同的基因将优先使用CRISPR/Cas9方法进行敲除。对于每个基因靶标，将设计两个gRNA对并注射到单细胞蝾螈胚胎中。胚胎将被饲养以评估生存能力，然后进行尾部截肢以确认CRISPR基因编辑并测试再生能力。与阻止或导致异常再生的CRISPR相关的胚胎将优先用于建立稳定的品系。Specific Aim 3将产生荧光报告细胞系，以通过已知在再生中起作用的主要途径测定信号传导活性，并且还将比较两种用于开发基因特异性报告细胞系的方法的功效，使用已知标记再生反应关键细胞群（神经干细胞，神经胶质细胞，肌肉卫星细胞）的测试基因座基因。化学和遗传的命中，以及从这个模型产生的生物信息将通过一个社区网站（Sal-Site）共享。拟议的转基因将由Ambystoma遗传储备中心分发。总的来说，该项目整合了化学筛选，药理学，组织学，转录，转基因和脊椎动物生物学的专业知识，以发现试剂并开发所需的工具，以增强美西螈的干细胞和再生生物学。