FSML: Single Cell Marine 'Omics at the Whitney Marine Lab for Bioscience

海洋环境拥有迄今为止地球上最大范围的生物多样性，这种生命多样性为了解生物如何“工作”以及它们如何与自然环境相互作用提供了尚未开发的机会。包括下一代DNA测序和功能基因组学在内的分子技术的最新发展为在发育、神经发生、癌症、适应性恢复和再生生物学过程中建立细胞调节的分子基础提供了新的认识水平。海洋动物，特别是无脊椎动物，是对动物基本特性进行各种研究的理想实验系统。该提案使用在惠特尼海洋生物科学实验室（http://www.whitney.ufl.edu/）收集和培养的精致海洋生物，利用这些新技术，在鉴定细胞的分辨率上对与基因表达有关的各种细胞行为进行前所未有的分子理解。该资助为分离鉴定的单细胞纯群体提供仪器，可用于下游分子表征（例如，构建涉及建立干细胞或神经身份的基因调控网络）。该设备将被安置在现场，并可以部署为广泛的正在进行的研究项目的驻地和访问研究人员。惠特尼实验室的所有研究小组都积极参与该实验室已有32年历史的NSF REU计划，参加K-12 STEM外展计划（例如科学家一天和惠特尼实验室的旅行动物园），3 - 4年级的夏令营，以及免费的公共讲座系列（“惠特尼之夜”）。惠特尼最近开设了一家海龟康复医院，专注于手术切除和治疗影响全球所有海龟物种的使人衰弱的纤维乳头状瘤（FP）疾病。惠特尼教师定期咨询社会和土地使用管理问题，并与当地社区分享知识和专业知识。该项目建立在以前的专业知识基础上，利用尖端的单细胞组学技术来了解不同细胞类型的分子调控。一个细胞标记设备（微注射装置），可以通过各种分子技术荧光标记单个细胞，以及一个高效的多用户友好的小体积荧光细胞分选器（BD FACSMelody）将被部署，以解决大量正在进行的研究项目的驻地和访问研究人员。荧光标记的细胞，无论是葡聚糖系标记物，DNA含量的核标记物，注射到活细胞中的特定基因的mrna，由内源性增强子驱动的报告基因，还是CRSPR/Cas9报告基因敲入蛋白，都可以分离成单个细胞，并使用FACS（荧光活化细胞分选机）进行分类。FACS机器曾经是昂贵的大型仪器，需要大量的细胞（1 × 106-9），只能对有限的波长通道进行分类。随着多个固态激光器和小体积分选器（96或384井格式）的发展，新一代的FACS机器变得更加强大。分离单细胞的能力与使用Next Gen测序技术对单个或少量分子鉴定细胞的转录组和基因组进行测序的能力是密切相关的。

Marine environments harbor by far the greatest range of biodiversity on Earth and this diversity of life provides untapped opportunities to understand how organisms "work" and how they interact with their natural environment. Recent developments in molecular technology in including next-generation DNA sequencing and functional genomics provide opportunities to build a new level of understanding of the molecular basis of cellular regulation during development, neurogenesis, cancer, adaptive resiliency, and regenerative biology. Marine animals, particularly invertebrates, are ideal experimental systems for a variety of investigations into fundamental properties of animals. This proposal uses delicate marine organisms, collected and cultured at the Whitney Lab for Marine Bioscience (http://www.whitney.ufl.edu/), to leverage these new technologies for an unprecedented molecular understanding of a variety of cellular behaviors related to the interrogation of gene expression at the resolution of identified cells. This grant provides instrumentation for the isolation of pure populations of identified single cells that can be used for downstream molecular characterization (e.g. the construction of gene regulatory networks involved in the establishment of stem cell or neural identity). This equipment will be housed on site, and can be deployed for a wide array of ongoing research projects of resident and visiting researchers. All research groups at the Whitney Lab remain actively involved in the Lab's 32 year-old NSF REU program, participate in K-12 STEM outreach programs (e.g. Scientist for a Day and Whitney Lab's Traveling Zoo), summer camps for 3rd- 4th graders, and a free public lecture series ("Evenings at Whitney"). Whitney recently opened a Sea Turtle Rehabilitation Hospital that focuses on the surgical removal and cure of debilitating fibropapilloma (FP) disease that affects all species of sea turtle worldwide. Whitney faculty are periodically consulted on social and land-use management issues, and share knowledge and expertise with the local community.This project builds on previous expertise to utilize cutting edge, single cell omics techniques to understand the molecular regulation of distinct cell types. A cell labeling facility (microinjection rig) that can fluorescently label individual cells via a variety of molecular techniques, and an efficient multiuser friendly small volume fluorescent cell sorter (BD FACSMelody) will be deployed to address a wide array of ongoing research projects of resident and visiting researchers. Fluorescently labeled cells, whether it is a dextran lineage marker, nuclear markers of DNA content, mRNAs for specific genes injected in to living cells, reporter genes driven by endogenous enhancers, or CRSPR/Cas9 reporter gene knock-ins can be dissociated into single cells and sorted using a FACS (Fluorescently Activated Cell Sorter) machine. FACS machines were once large prohibitively expensive instruments needing large numbers of cells (1 x 106-9 ) and only able to sort limited wavelength channels. Newer generation FACS machines have become much more powerful with the development of multiple solid state lasers and small volume sorters (96 or 384 well formats). The capability to isolate single cells goes hand-in-hand with the ability to sequence transcriptomes and genomes of single or small numbers of molecularly identified cells, using Next Gen sequencing technology.