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(由申请人提供)：这个多PD/PI应用程序寻求开发资源，以更好地支持对高度再生的两栖动物墨西哥Axolotl(Ambystoma Micianum)的研究。有必要更深入地探索Axolotl的再生，使用在遗传模式生物中被证明是如此有效的同样强大的方法。具体目标1将利用胚胎尾部再生试验完成第一次紫杉醇再生的化学遗传筛选。使用在微量培养皿中高效饲养的预喂养Axolotl胚胎，将测试来自商业、临床阶段和新型天然产品库的多达10,000种可溶化学物质对尾巴再生的影响。初步数据显示，化学筛选和尾巴再生试验很可能识别出影响再生的新分子。来自该筛查的阳性结果，包括先前确定的Wnt、转化生长因子β和成纤维细胞生长因子信号的抑制物，将被报告给社区，并在特定的目标2下，使用细胞和转录方法进行进一步的研究。特别是，将开发分析方法来评估创面表皮形成的化学效应，创面表皮在祖细胞/干细胞招募中扮演早期信号中心的角色。此外，还将开发检测方法来评估细胞增殖和去分化，这两个过程与内源性再生有关。在对照和化学处理的胚胎中发现表达不同的基因将使用CRISPR/Cas9方法优先敲除。对于每个基因目标，将设计两对gRNA并将其注射到单细胞Axolotl胚胎中。胚胎将被饲养以评估存活能力，然后进行尾部截肢以确认CRISPR基因编辑并测试再生能力。与CRISPR相关的胚胎如果阻碍或导致异常再生，将优先建立稳定的品系。特殊目标3将产生荧光报告系，以通过已知在再生中起作用的主要途径来分析信号活性，并将比较两种开发基因特异性报告系的方法的效率，使用已知的标记对再生反应至关重要的细胞群(神经干细胞、神经胶质细胞、肌肉卫星细胞)的基因作为测试者基因座。这一模式产生的化学和遗传信息以及生物信息将通过一个社区网站(SAL-SITE)共享。拟议的转基因药物将由Ambystoma基因库存中心分发。总体而言，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方面的专业知识，以发现试剂并开发必要的工具，以增强干细胞和再生生物学中的axolotl。