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.

这项提议试图建立一种在模式生物体中测试引发肢体再生的因素， 这将为设计肌肉、骨骼结构甚至整体的方法提供参考 人类的四肢才能再生。非洲爪哇蝌蚪能在早期再生四肢 处于发育阶段，但不是在后期。当前提议的理由是任何可再生的 机制丢失可能在哺乳动物的四肢中缺失，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。精挑细选的 系统将分离已适当重新指定的细胞，并对再生的 肌纤维、离散的肌肉和整个再生的附件将用 肌肉力量评估核心的帮助。此项目使用的方法很少(如果有的话)被用于 研究肌肉发育、分化和再生：iPS技术重新定义细胞；组织培养 方法研究再生情况，用流式细胞仪选择所需细胞。《公约》的具体目标 建议包括： 特异目的1：将弗罗格莱特肢源性成纤维细胞重新分化为肢芽状态。工作假说 这可以通过引入编码肢芽状态的基因，以及 打开染色质，然后对阳性反应的细胞进行选择性步骤。 特定目标2：通过重新引入肢芽来挽救非洲爪蛙肢体的再生能力 细胞。工作假设是，这可以通过皮下注射肢芽细胞来实现， 允许愈合，并通过移植物区域截肢。