Developing Regeneration Resources for a Model Amphibian

为模型两栖动物开发再生资源

• 批准号: 9072228
• 负责人: Jon Scott Thorson
• 金额: $ 65.79万
• 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/9072228
• 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; Epidermis; Event; FDA approved; Future; Gene Expression; Gene Targeting; Gene Transfer Techniques; Genes; Genetic; Genetic Models; Genetic Screening; Genetic Transcription; Guide RNA; Histology; Hormone use; Information Resources; Insertional Mutagenesis; Investigation; Knock-out; Libraries; Life; Maps; Mediating; Methods; Modeling; Molecular; Molecular Profiling; Monitor; Muscle satellite cell; Mutagenesis; 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; Signaling Protein; Site; Somatic Cell; Staging; Stem Cell Research; Stem cells; Tail; Testing; Thyroid Hormones; Time; Tissues; 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

 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（由应用程序提供）：此多PD/PI应用程序旨在开发资源，以更好地研究高度再生两栖动物的墨西哥Axolotl（墨西哥ambystoma）。有必要更深入地探测Axolotl的再生，具有相同强大的方法在遗传模型生物中如此有效。特定的目标1将使用胚胎尾再生测定法完成第一个化学遗传筛选。使用有效饲养在微孔板上的预喂养的Axolotl胚胎，将测试来自商业，临床阶段和新型天然产品库中多达10,000种固体化学物质，以影响尾部再生。初步数据表明，化学筛选和尾部再生测定法可能会确定影响再生的新分子。该屏幕的正命中率，包括先前鉴定的Wnt，TGFβ和FGF信号传导的抑制剂，并将使用细胞和转录方法在特定的目标2下进行进一步研究，并在特定的目标2下进行进一步研究。特别是，将开发分析以评估对伤口表皮形成的化学作用，该化学对祖细胞/干细胞的募集中的早期信号传导中心。同样，将开发分析以评估细胞增殖和分化，这是与内源性再生有关的两个过程。在对照和化学处理的胚胎之间发现的基因表达不同的基因将优先使用CRISPR/CAS9方法来淘汰。对于每个基因靶标，将设计两个GRNA对并注入单细胞Axolotl胚胎。将饲养胚胎以评估生存能力，然后施用尾部截肢，以确认CRISPR基因编辑并测试再生能力。与CRISPR相关的胚胎将优先考虑阻断或引起异常再生的稳定线。特定的目标3将通过已知在再生中功能的主要途径来生成荧光报告信号传导活性，还将比较开发基因特异性报告基因线的两种方法的效率，该方法使用已知的局部基因已知用于标记细胞群体对再生反应至关重要的细胞群体（神经干细胞，神经干细胞，肌肉，肌肉卫星细胞）。该模型产生的化学和遗传热门以及生物学信息将通过社区网站（SAL SITE）共享。所提出的转换将由Ambystoma遗传储备中心分布。总体而言，该项目将化学筛查，药理学，组织学，转录，转基因和脊椎动物生物学的专业知识融为一体，以发现试剂和开发所需的工具，以增强干细胞和再生生物学。