EDGE CT: Developing transgenic and lineage tracing tools in planarians

EDGE CT：开发涡虫转基因和谱系追踪工具

• 批准号: 1923534
• 负责人: Bo Wang
• 金额: $ 50万
• 依托单位: Stanford University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1923534&HistoricalAwards=false
• 关键词: EDGE; CT; Developing; transgenic; lineage

Planarians have captured the imagination of generations of scientists, and today play a critical role in our efforts to understand development and regeneration. These small flatworms can regenerate their entire body from small tissue fragments. As a result of the current surge in the sequencing technology, tremendous progress has been made over the past two decades to understand planarian biology from a variety of perspectives, including but not limited to regeneration, stem cell, animal body plan and evolution, germline, nervous system, metabolism, innate immunity, signaling pathways, regulatory RNAs, and genome stability. Yet the entire field has been limited by the lack of tools for transgene expression and genome editing. At the same time, technological innovation has exploded for transfection and gene editing of mammalian cells. In particular, nanotechnological approaches can very locally disrupt the cell membrane and inject molecular cargo directly, providing both low cell toxicity and high delivery efficiencies. In this project, the researchers will use the novel nanotechnology, "nanostraws," to deliver genetic materials into planarian cells, and provide a simple and effective means for transgene expression and genome editing. This project also aims to rapidly disseminate the new technology by facilitating data, protocol, and materials sharing across labs, and organizing training workshops. In addition, because of planarians' appealing regenerative ability, childlike cuteness, and ease to rear in the lab, the researchers will promote the usage of planarians in teaching, education, and outreach activities to engage non-scientists' interests in modern biological research.Planarians have been a powerful animal model to study tissue regeneration and stem cell biology. They have the unique capacity to regenerate the entire body from minute tissue fragments using pluripotent somatic stem cells. During this process, they reset the body axes and rebuild all organs in appropriate proportions. Although extensive genomic and transcriptomic information is available, progress on addressing causal genotype-phenotype relationships in planarians has been severely hindered by a lack of transgenic tools despite decades of attempts. Enabled by the recent technical advances made in the investigators' laboratories, including cell culture and transplantation methods, novel nanotechnology for gene delivery in hard-to-transfect cell types, and successful expression of luminescence reporters in planarian cells, they aim to address this long-standing challenge. This research project aims to develop and disseminate the methodology and resources for planarian transgenesis. Specifically, the goals of this project include (1) to develop nanoscale electroporation methods for transgene delivery in planarian cells, (2) to demonstrate genome editing using CRISPR/Cas9, and (3) to enhance the collaboration, training, and diversity of the planarian research community to address a broad spectrum of questions in molecular, cellular, organismal, and evolutionary biology.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Planarians吸引了几代科学家的想象力，今天在我们理解发展和再生的努力中发挥着关键作用。这些小扁形虫可以从小的组织碎片中再生出整个身体。由于目前测序技术的激增，在过去二十年中已经取得了巨大的进步，从各种角度来理解涡虫生物学，包括但不限于再生，干细胞，动物身体计划和进化，生殖系，神经系统，代谢，先天免疫，信号传导途径，调节RNA和基因组稳定性。然而，由于缺乏转基因表达和基因组编辑的工具，整个领域受到了限制。与此同时，哺乳动物细胞的转染和基因编辑技术创新也出现了爆炸式增长。特别是，纳米技术方法可以非常局部地破坏细胞膜并直接注入分子货物，从而提供低细胞毒性和高递送效率。在该项目中，研究人员将利用新型纳米技术“纳米吸管”将遗传物质输送到真涡虫细胞中，并为转基因表达和基因组编辑提供简单有效的手段。该项目还旨在通过促进实验室之间的数据，协议和材料共享以及组织培训研讨会来快速传播新技术。此外，由于真涡虫具有吸引人的再生能力，孩子般的可爱，以及易于在实验室中饲养，研究人员将在教学，教育和推广活动中推广真涡虫的使用，以吸引非科学家对现代生物学研究的兴趣。真涡虫已经成为研究组织再生和干细胞生物学的强大动物模型。他们有独特的能力，再生整个身体从微小的组织碎片使用多能体干细胞。在这个过程中，他们重置身体轴，并以适当的比例重建所有器官。虽然广泛的基因组和转录组学信息，解决因果基因型-表型关系的涡虫的进展已严重阻碍了缺乏转基因工具，尽管几十年的尝试。通过研究人员实验室最近取得的技术进步，包括细胞培养和移植方法，用于难以检测的细胞类型中基因递送的新型纳米技术，以及在Planarian细胞中成功表达发光报告基因，他们的目标是解决这一长期挑战。该研究项目旨在开发和传播真涡虫转基因的方法和资源。具体而言，该项目的目标包括（1）开发用于在真涡虫细胞中进行转基因递送的纳米级电穿孔方法，（2）展示使用CRISPR/Cas9的基因组编辑，以及（3）加强真涡虫研究社区的合作，培训和多样性，以解决分子，细胞，生物，该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。