Use of specific transcription factors to promote limb regeneration capacity

本发明的目的是建立一种在模式生物体中引起肢体再生的因素的测定方法， 这将为设计方法的平行努力提供信息， 人类的四肢得以再生非洲爪蟾蝌蚪在早期就能再生肢体 发展阶段，但不是后期阶段。目前提案的基本原理是， 哺乳动物的四肢可能缺少一种机制，这是它们无法再生的原因： 肢体成纤维细胞可以通过特异性转录因子重新指定为肢体芽表型。选择性 系统将分离已适当重新指定的细胞，并再生细胞的性能 肌纤维、离散肌肉和整个再生附肢将用 肌肉力量评估核心的帮助。这个项目使用的方法很少，如果有的话，被用来 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 建议如下： 具体目的1：将蛙肢来源的成纤维细胞重新鉴定为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来实现， 展开染色质，然后进行阳性反应细胞的选择步骤。 具体目标2：通过重新引入肢芽来挽救爪蟾幼蛙肢的再生能力 细胞目前的假设是，这可以通过皮下注射肢芽细胞来实现， 允许愈合，并通过移植区域进行截肢。

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.