EDGE FGT: Creation of a Genetically Tractable Cephalopod Model using the Hummingbird Bobtail Squid

头足类动物为生物学发现提供了巨大的机会。它们的新奇之处包括最复杂的无脊椎动物神经系统和行为，创造近乎完美的伪装的能力，独特的身体结构，分类群特定的基因和基因家族扩展，宿主微生物的专门器官，以及在mRNA中编辑遗传信息的巨大能力。在神经科学、发育生物学、生理学、生物力学、微生物-宿主相互作用、材料特性、基因组学和分子生物学等领域，有一个庞大而多样的头足类研究群体。尽管有这些生物学特性，头足类动物的研究一直受到限制，因为很难获得标本，而且完全缺乏遗传上可处理的模式物种。为此，我们正在为蜂鸟短尾鱿鱼（Euprymna berryi）开发基因操作和饲养方法和资源，这是一种原产于日本的物种，在实验室环境中已被证明是健壮的。作为这项工作的一部分，我们将开发驱动报告基因表达的技术，并利用这些方法创建转基因品系和资源，为研究界服务。在这项工作中开发的工具和方法将为研究头足类生物的庞大和多样化的社区开辟新的研究领域，我们将举办以实验室为基础的实践研讨会，以迅速分享我们的技术进步。通过这个项目，我们还创建了一个实习项目，提供指导、专业发展和在职培训，帮助高水平的学生从事水产养殖者和水生生物研究人员的职业。头足类生物研究的一个关键瓶颈是缺乏一种遗传上可处理的模式物种。这在一定程度上是由于在实验室环境中培养头足类动物相对困难，难以获取和操纵早期胚胎阶段，以及缺乏该分类单元的分子和测序资源。为了解决这些问题，我们的目标是开发出一种培育短尾鱿鱼的转基因株系，并利用这些方法创造资源为研究界服务。作为这项工作的一部分，我们将通过测试DNA磁带的随机和特定位点插入来优化创建转基因的协议。我们还将创建序列资源，用于鉴定启动子和增强子，以驱动组织特异性和普遍存在的报告基因表达。最后，我们将建立健全的饲养条件，以维持这些品种的多代繁殖。这项工作预计将对头足类研究界产生重大影响，提供新的工具和技术，开辟新的研究途径，使新的和已建立的研究实验室都能接触到头足类生物学。该奖项反映了美国国家科学基金会的法定使命，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。

Cephalopods offer tremendous opportunities for biological discovery. Their novelties include the most complex invertebrate nervous systems and behaviors, the ability to create near-perfect camouflage, unique body plans, taxon specific genes and gene family expansions, specialized organs for hosting microbes, and an enormous capacity to edit genetic information within mRNA. There is a large and diverse cephalopod research community that takes advantage of these unique features for neuroscience, developmental biology, physiology, biomechanics, microbial-host interactions, material properties, genomics, and molecular biology. Despite these biological attributes, cephalopod research has been limited because specimens are difficult to obtain and there is a complete lack of genetically tractable model species. To this end, we are developing gene manipulation and husbandry methods and resources for Euprymna berryi, the hummingbird bobtail squid, a species native to Japan that has proven to be robust in laboratory settings. As part of this effort, we will develop the technologies to drive reporter gene expression and use these methods to create transgenic lines and resources to serve the research community. The tools and methods developed in this work will open up new areas of investigation for the large and diverse community that studies cephalopod biology, and we will hold laboratory-based hands-on workshops to rapidly share our technical advances. Through this program, we have also created an internship program to provide mentorship, professional development, and on-the-job training to aid upper-level students pursuing careers as aquarists and researchers using aquatic organisms. A key bottleneck in the study of cephalopod biology is the absence of a genetically tractable model species. This is, in part, due to the relative difficulty of culturing cephalopods in laboratory settings, the difficulty of accessing and manipulating early embryonic stages, and the absence of molecular and sequencing resources for this taxon. To address this these limitations, our goal is to develop the methods to create transgenic lines of Euprymna berryi, a culturable bobtail squid, and then to use these methods to create resources to serve the research community. As part of this effort, we will optimize protocols for creating transgenics by testing random and site-specific insertion of DNA cassettes. We will also create sequence resources for the identification of promoters and enhancers to drive tissue-specific and ubiquitous reporter gene expression. Finally, we will establish robust husbandry conditions for maintaining these lines across multiple generations. This work is expected to have a strong impact on the cephalopod research community by providing new tools and techniques that will open novel avenues of inquiry, making cephalopod biology accessible to both new and established research labs.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.