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

海洋环境拥有地球上迄今为止最大范围的生物多样性，这种生物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。 分子技术的最新发展，包括下一代DNA测序和功能基因组学，为建立对发育、神经发生、癌症、适应性恢复和再生生物学过程中细胞调节的分子基础的新水平的理解提供了机会。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。该提案使用在惠特尼海洋生物科学实验室（http：//www.whitney.ufl.edu/）收集和培养的脆弱海洋生物，利用这些新技术对与在识别细胞的分辨率下询问基因表达相关的各种细胞行为进行前所未有的分子理解。该资助提供了用于分离经鉴定的单细胞纯群体的仪器，这些单细胞可用于下游分子表征（例如，参与建立干细胞或神经身份的基因调控网络的构建）。这些设备将安置在现场，并可用于驻地和访问研究人员正在进行的各种研究项目。 惠特尼实验室的所有研究小组仍然积极参与实验室32年的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年级学生夏令营，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衰弱性纤维乳头状瘤（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。一个细胞标记设施（显微注射装置），可以通过各种分子技术荧光标记单个细胞，和一个高效的多用户友好的小体积荧光细胞分选仪（BD FACSMelody）将被部署，以解决居民和访问研究人员正在进行的研究项目的广泛阵列。经免疫标记的细胞，无论是葡聚糖谱系标记物、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或CRSPR/Cas9报告基因敲入，都可以解离成单细胞并使用FACS（免疫激活细胞分选仪）机器分选。流式细胞仪曾经是非常昂贵的大型仪器，需要大量的细胞（1 × 106-9），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体积分选机（96或384孔格式）的发展，新一代的FACS机器已经变得更加强大。分离单细胞的能力与使用下一代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密切相关。

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.