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

到目前为止，海洋环境中蕴藏着地球上最大范围的生物多样性，这种生命的多样性提供了尚未开发的机会，以了解有机体如何“工作”，以及它们如何与自然环境相互作用。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建立对发育、神经发生、癌症、适应性弹性和再生生物学过程中细胞调控的分子基础的理解提供了机会。海洋动物，特别是无脊椎动物，是研究动物基本特性的理想实验系统。这项计划使用惠特尼海洋生物科学实验室(http://www.whitney.ufl.edu/)，)收集和培养的脆弱海洋生物，利用这些新技术对各种细胞行为进行前所未有的分子理解，这些行为与在已识别细胞的分辨率下询问基因表达有关。这笔赠款为分离可用于下游分子鉴定的已鉴定单个细胞的纯群体提供了工具(例如，构建参与建立干细胞或神经身份的基因调控网络)。这些设备将被安置在现场，并可部署在常驻和访问研究人员的广泛正在进行的研究项目中。惠特尼实验室的所有研究小组仍然积极参与该实验室已有32年历史的NSF REU计划，参与K-12 STEM外联计划(例如，科学家一天和惠特尼实验室的巡回动物园)，为3-4年级的学生举办夏令营，以及免费的公共讲座系列(“晚上在惠特尼”)。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种类海龟的衰弱纤维乳头状瘤(FP)疾病。惠特尼学院定期就社会和土地使用管理问题进行咨询，并与当地社区分享知识和专业知识。该项目以以前的专业知识为基础，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。将部署可通过各种分子技术对单个细胞进行荧光标记的细胞标记设备(微注射装置)，以及高效的多用户友好的小体积荧光细胞分选器(BD FACSMelody)，以满足常驻和访问研究人员正在进行的一系列研究项目。荧光标记的细胞，无论是葡聚糖谱系标记、DNA含量的核标记、注入活细胞的特定基因的mRNAs、内源性增强子驱动的报告基因，还是CRSPR/Cas9报告基因敲入，都可以分离到单个细胞中，并使用FACS(荧光激活细胞分类器)机器进行分选。FACS机曾经是大型的、昂贵的仪器，需要大量的细胞(1x106-9)，并且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容量分选机(96或384孔格式)的发展，新一代FACS机变得更加强大。分离单个细胞的能力与使用下一代测序技术对单个或少量分子识别的细胞的转录和基因组进行测序的能力是齐头并进的。

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.