A genetic system for the study of vertebrate limb regeneration

描述(由申请人提供)：在再生医学的广泛框架内，肌肉骨骼再生提出了特别的挑战。虽然干细胞生物学和组织工程学的进步使我们更接近能够产生具有生理功能的组织的目标，包括软骨、骨骼、肌肉和肌腱，但肌肉骨骼系统的功能需要这些组织相互组织和整合，并与神经系统成功发挥作用。因此，尽管增强局部肌肉或骨骼损伤修复的能力一直在提高，并可能在未来以更快的速度继续这样做，但在更灾难性的损失后再生功能性肌肉骨骼系统的能力仍然是一个遥远的梦想。在这一关键领域进展如此之少的一个主要原因是缺乏解决这一问题的易驯服的动物模型。虽然斑马鱼可以再生它们的鳍，并且非常适合于分子和遗传分析，但能够再生的鳍部分(鳍射线)既不包含软骨内骨，也不包含肌肉和肌腱。因此，唯一一类能够再生整个肌肉骨骼结构的脊椎动物是尾目两栖动物、火蜥蜴及其近亲。然而，由于缺乏实验工具，两栖动物肢体再生的研究一直处于相对停滞的状态。利用Axolotl，一种非常适合在实验室环境中使用的火蜥蜴，这项提议是开发方法和转基因动物，将允许在现代基因水平上解决肢体再生的问题。这将包括创建一个特定于组织的基因活动操纵系统(以便可以评估基因在特定再生组织中的作用)，用于有条件地调节基因表达(使得肢体的发育可以正常进行，并且只能在再生过程的特定阶段启动基因激活或敲除)。此外，除了在转基因动物中进行明确的基因操作外，还将建立利用病毒载体快速高效地改变基因表达的方法。建立这样的遗传系统将对未来在高等生物体中再生综合肌肉骨骼系统的工作产生变革性的影响。 公共卫生相关性：该项目的目标是开发一种遗传系统，用于研究完整的肌肉骨骼系统的再生，例如在肢体中发现的肌肉骨骼系统。在Axolotls中的转基因方法将被用来开发一种具有组织特异性和时间控制的激活和敲除基因功能的方法。

DESCRIPTION (provided by applicant): Within the broad framework of regenerative medicine, musculoskeletal regeneration poses particular challenges. While advances in stem cell biology and tissue engineering have brought us closer to the goal of being able to produce physiologically competent tissues, including cartilage, bone, muscle and tendons, the function of the musculoskeletal system requires that these tissues be organized and integrated with one another and with the nervous system to function successfully. Thus, while the ability to enhance repair of local muscle or skeletal damage is improving all the time and will likely continue to do so at an accelerating rate in the future, the capacity to regenerate a functional musculoskeletal system after more catastrophic loss remains a distant dream. A major reason that there has been so little progress in this critical area has been the lack of a tractable animal model for addressing it. While zebrafish can regenerate their fins and are well suited to molecular and genetic analyses, the portion of the fin capable of regenerating (the fin rays) contains neither endochondral bone nor muscle and tendons. Hence, the only class of vertebrates capable of regenerating entire musculoskeletal structures is the urodele amphibians, the salamanders and their relatives. However, research on amphibian limb regeneration has remained a relative backwater due to the lack of experimental tools. Utilizing the axolotl, a salamander that is highly suited for use in a laboratory setting, this proposal is to develop methods and transgenic animals that will allow the problem of limb regeneration to be addressed on a modern genetic level. This will include creating a system for tissue-specific manipulation of gene activity (such that the roles of genes in specific regenerating tissues can be assessed), for conditionally regulating gene expression (such that development of the limb can occur normally and gene activation or knock-down can be initiated only during specific stages of the regenerative process). Moreover, methods will be established for rapidly altering gene expression at a high efficiency with viral vectors in addition to definitive gene manipulation in transgenic animals. Establishing such a genetic system will be transformative for future work towards regenerating an integrative musculoskeletal system in higher organisms. PUBLIC HEALTH RELEVANCE: The goal of this project is to develop a genetic system for studying the regeneration of integrated musculoskeletal systems such as is found in the limb. Transgenic approaches in axolotls will be utilized to develop a means of activating and knocking down gene function with tissue-specificity and temporal control.