Use of specific transcription factors to promote limb regeneration capacity

利用特定转录因子促进肢体再生能力

• 批准号: 7685935
• 负责人: JOHN W DAY
• 金额: $ 8.18万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7685935
• 关键词: Amphibia; Amputation; Animal Model; Biological Assay; Body part; Cell Differentiation process; Cells; Chromatin; Development; Developmental Biology; Disease; Fibroblasts; Flow Cytometry; Gene Targeting; Genes; Goals; Healed; Health; Human; Knowledge; Laboratories; Limb Bud; Limb structure; Methods; Muscle; Muscle Development; Muscle Fibers; Muscular Dystrophies; Musculoskeletal; Mutation; Myopathy; Natural regeneration; Performance; Phenotype; Pilot Projects; Process; Rana; Research; Skeletal Muscle; Specific qualifier value; Staging; Stem cells; System; Tadpoles; Technology; To specify; Work; Xenopus; Xenopus laevis; appendage; design; healing; induced pluripotent stem cell; limb regeneration; regenerative; stem cell technology; tissue/cell culture; transcription factor

This proposal seeks to establish an assay for factors provoking limb regeneration in a model organism, which will inform parallel efforts to design methods for enabling muscle, skeletal structures or even whole limbs of human beings to regenerate. Tadpoles of Xenopus laevis can regenerate limbs at early developmental stages but not at later stages. The rationale for the current proposal is whatever regenerative mechanism is lost is probably missing in mammalian limbs and responsible for their inability to regenerate: limb fibroblasts can be re-specified to a limb bud phenotype by specific transcription factors. A selective system will isolate cells that have been appropriately re-specified, and performance of the regenerated muscle fibers, discrete muscles, and entire regenerated appendages will be assessed quantitatively with the help of the Muscle Force Assessment Core. This project uses methods that have seldom, if ever, been used to study muscle development, differentiation and regeneration: iPS technology to re-define cells; tissue culture methods to investigate regeneration; use of flow cytometry to select the desired cells. Specific Aims of the proposal are: Specific Aim 1; To respecify froglet limb-derived fibroblasts to a. limb bud state. The working hypothesis is that this can be achieved by introduction of genes encoding the limb bud state, together with genes to unwrap the chromatin, followed by a selective step for positively responding cells. Specific Aim 2: To rescue regenerative ability in Xenopus froglet limbs by re-introduction of limb bud cells. The working hypothesis is that this can be achieved by injecting limb bud cells subcutaneously, allowing to heal, and amputation through the graft region.

该提案旨在建立一种在模型生物体中引发肢体再生的因素的测定方法， 这将为并行努力提供设计方法，使肌肉、骨骼结构甚至整个 使人类的四肢得以再生。非洲爪蟾蝌蚪可早期再生四肢 发育阶段，但不是后期。当前提案的基本原理是再生性的 哺乳动物四肢中可能缺少机制丢失，并导致它们无法再生： 肢体成纤维细胞可以通过特定的转录因子重新指定为肢芽表型。有选择性的 系统将隔离已适当重新指定的细胞，以及再生的性能 肌纤维、离散肌肉和整个再生附肢将通过 肌肉力量评估核心的帮助。该项目使用了很少（如果有的话）被使用过的方法 研究肌肉发育、分化和再生：iPS技术重新定义细胞；组织培养 研究再生的方法；使用流式细胞术选择所需的细胞。具体目标 提案是： 具体目标1；将青蛙肢体来源的成纤维细胞重新指定为a。肢芽状态。工作假设 这可以通过引入编码肢芽状态的基因以及 打开染色质，然后对阳性反应细胞进行选择性步骤。 具体目标 2：通过重新引入肢芽来恢复爪蟾四肢的再生能力 细胞。工作假设是，这可以通过皮下注射肢芽细胞来实现， 允许通过移植区域愈合和截肢。