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.

平面主义者抓住了几代科学家的想象，今天在我们了解发展和再生的努力中起着至关重要的作用。这些小的扁虫可以从小组织碎片中再生其整个身体。由于目前的测序技术激增，在过去的二十年中取得了巨大的进步，以从多种角度理解Planarian生物学，包括但不限于再生，干细胞，动物身体计划和进化，种系，神经系统，代谢系统，天生的免疫，先天免疫，信号途径，调节性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工具，整个领域受到了限制。同时，技术创新已经爆炸，用于转染和基因编辑的哺乳动物细胞。特别是，纳米技术方法可以在非常局部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，研究人员将使用新型的纳米技术“纳米丝”将遗传材料传递到平层细胞中，并为转基因表达和基因组编辑提供一种简单有效的手段。该项目还旨在通过促进跨实验室的数据，协议和材料共享并组织培训研讨会来快速传播新技术。此外，由于平面主义者具有吸引人的再生能力，幼稚的可爱性以及在实验室中易于培养，研究人员将促进平面人在教学，教育和外展活动中的使用，以吸引非科学家在现代生物学研究中的利益。Planararians在研究组织和干细胞生物学方面一直是一种强大的动物模型。它们具有独特的能力，可以使用多能体细胞从微小组织碎片中再生整个身体。在此过程中，它们以适当的比例重置身体轴并重建所有器官。尽管有广泛的基因组和转录组信息可用，但是尽管尝试了数十年的尝试，但由于缺乏转基因工具而严重阻碍了平面群体中的因果基因型 - 表型关系的进展。由研究人员实验室中最近的技术进步（包括细胞培养和移植方法），难以转移的细胞类型中基因递送的新型纳米技术以及在Planarian细胞中的发光记者的成功表达，他们旨在应对这一长期存在的挑战。该研究项目旨在开发和传播Planarian转基因的方法和资源。具体而言，该项目的目标包括（1）开发纳米级电穿孔方法，用于在Planarian细胞中传递转基因，（2）使用CRISPR/CAS9证明基因组编辑，以及（3）增强Planarian研究社区的协作，培训和多样性，以解决分子，细胞，生物体统计和进化态度，并评估广泛的问题，以解决广泛的问题。认为值得通过基金会的智力优点和更广泛影响的评论标准来评估值得支持。

项目成果

Mechanical expansion microscopy

• DOI: 10.1016/bs.mcb.2020.04.013
• 发表时间: 2021-01-01
• 期刊: EXPANSION MICROSCOPY FOR CELL BIOLOGY
• 作者: Fan, Yuhang;Lim, Youngbin;Wang, Bo
• 通讯作者: Wang, Bo