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

Cephalos豆荚为生物发现提供了巨大的机会。他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，独特的身体计划，分类特异性基因和基因家族扩展，宿主微生物的专门器官，以及编辑mRNA中遗传信息的巨大能力。有一个庞大而多样的头足类动物研究社区，利用这些独特的功能，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。尽管有这些生物学特性，但头足类动物的研究一直受到限制，因为标本很难获得，而且完全缺乏遗传学上易处理的模式物种。为此，我们正在为Euprymna berryi（蜂鸟短尾鱿鱼）开发基因操作和饲养方法和资源，Euprymna berryi是日本本土的一种物种，已被证明在实验室环境中具有强大的生命力。作为这项工作的一部分，我们将开发技术来驱动报告基因的表达，并使用这些方法来创建转基因品系和资源，以服务于研究界。在这项工作中开发的工具和方法将为研究头足类生物学的大型和多样化的社区开辟新的调查领域，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。通过这个计划，我们还创建了一个实习计划，提供指导，专业发展和在职培训，以帮助高层次的学生追求事业作为水族馆和研究人员使用水生生物。 头足类生物学研究的一个关键瓶颈是缺乏一个遗传上易处理的模式物种。这部分是由于在实验室环境中培养头足类动物的相对困难，访问和操纵早期胚胎阶段的困难，以及缺乏此分类群的分子和测序资源。为了解决这些限制，我们的目标是开发方法来创建可培养的短尾乌贼Euprymna berryi的转基因品系，然后利用这些方法来创建资源，为研究界服务。作为这项工作的一部分，我们将通过测试DNA盒的随机和位点特异性插入来优化创建转基因的方案。我们还将创建序列资源，用于识别启动子和增强子，以驱动组织特异性和普遍存在的报告基因表达。最后，我们将建立强大的饲养条件，以保持这些线跨多代。这项工作有望通过提供新的工具和技术，开辟新的研究途径，使头足类动物生物学进入新的和建立的研究实验室，对头足类动物研究界产生强烈的影响。该奖项反映了NSF的法定使命，并已被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

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.