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IOS EDGE: Nanoscale Probes and Infrastructure for Real-Time and Single-Cell Genomics across Metazoa

IOS EDGE：用于后生动物实时和单细胞基因组学的纳米级探针和基础设施

基本信息

批准号：
1645219
负责人：
Leonid Moroz
金额：
$ 100万
依托单位：
University of Florida
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2017
资助国家：
美国
起止时间：
2017-07-15 至 2021-06-30
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1645219&HistoricalAwards=false
关键词：
IOS EDGE Nanoscale Probes Infrastructure

项目摘要

The genetic material, or genome, first and foremost operates at the level of specific cells, and practically any animal tissue or embryo consists of thousands of highly diverse cells. How and why the same genome leads to such enormous diversity of cell types and functions are unanswered questions of modern biology. Yet, cell-specific approaches to link cause and effect are virtually absent for a majority of animal groups. This interdisciplinary project addresses these bottlenecks experimentally by developing novel genomic approaches and chemical labeling tools for genome-wide characterization of expression, classification, and mapping of thousands of individual cells in parallel. This information is used to (i) achieve a nearly complete census of cell types within a given organism, focusing on animal models critical to understanding mechanisms of learning and memory, such as Aplysia, and regeneration, such as Pleurobrachia, and (ii) generate nanoscale probes that selectively mark specific cells for genome editing, regardless of any advance knowledge about the cells' molecular diversity. Several communities are benefiting from the proposed research, including comparative neurobiology, development, biological oceanography, and the emerging field of synthetic biology. The project also affords cross-disciplinary training opportunities for trainees from the undergraduate to postdoctoral level and educational outreach activities in marine and comparative biology aimed at a diverse K-12 student body. The grand challenge in our understanding of the genomes-to-phenomes relationships is our general inability to manipulate genome operation at the level of specific individual cells at any given location and at any given time. These obstacles are more dramatic for most invertebrates, when researchers study development or neuronal functions with little information about the cellular composition of target organs. Here, microfluidics for massive parallel single-cell capture and sequencing are integrated with novel cell selection technologies, such as aptamer-based-Cell-SELEX, for quantitative gene expression analyses and imaging of individual cells in intact tissues. Aplysia (and, once single-cell tools are validated, Pleurobrachia and/or related ctenophore species) are used to achieve nearly complete genome-wide classification of the majority of cell types in their neural systems and effector organs. The read-out(s) to measure/control gene expression in identified neurons are: scRNA-seq data with both normalized and absolute quantification of expression levels for target genes, and q-RT-PCR. Controls are neurons in which target genes are not active or silenced. First, unique resources for a diversity of cell adhesion molecules and other surface macromolecular structures critical to design and characterize cell-specific probes are generated. Then, using tools of chemical evolution, a high-throughput system to manufacture cell-specific aptamer-/molecular beacon-based fluorescent probes at a large scale is tested. Finally, hybrid nanoscale probes (e.g. made by coupling cell-specific fluorescent markers with nucleic acid analogues) are tested for their ability to self-deliver molecular constructs into target cells without direct injection, electroporation, or the need to make transgenic animals. This project is co-funded by the Chemistry of Life Processes program in the Division of Chemistry.

遗传物质或基因组首先在特定细胞水平上起作用，实际上任何动物组织或胚胎都由数千个高度多样化的细胞组成。相同的基因组如何以及为什么会导致细胞类型和功能如此巨大的多样性，这是现代生物学中没有答案的问题。然而，对于大多数动物群体来说，细胞特异性的方法来联系因果关系实际上是缺失的。这个跨学科项目通过开发新的基因组方法和化学标记工具来解决这些实验瓶颈，这些方法和化学标记工具可以并行地对数千个单个细胞的表达、分类和定位进行全基因组表征。这些信息用于(i)在给定生物体中实现几乎完整的细胞类型普查，重点关注对理解学习和记忆机制至关重要的动物模型，如海雀，以及再生，如胸膜鱼，以及（ii）产生纳米级探针，选择性地标记特定细胞进行基因组编辑，而不考虑任何关于细胞分子多样性的预先知识。包括比较神经生物学、发展、生物海洋学和新兴的合成生物学领域在内的几个社区正从拟议的研究中受益。该项目还为从本科生到博士后水平的受训者提供跨学科培训机会，并为不同的K-12学生群体提供海洋和比较生物学方面的教育推广活动。在我们理解基因组-现象关系的过程中，最大的挑战是我们通常无法在任何给定地点和任何给定时间在特定单个细胞的水平上操纵基因组操作。对于大多数无脊椎动物来说，当研究人员研究发育或神经元功能时，对目标器官的细胞组成知之甚少，这些障碍就更加明显了。在这里，用于大规模平行单细胞捕获和测序的微流体与新型细胞选择技术相结合，例如基于适配体的cell - selex，用于定量基因表达分析和完整组织中单个细胞的成像。我们使用applysia（一旦单细胞工具得到验证，也可以使用Pleurobrachia和/或相关的栉水母物种）来实现其神经系统和效应器官中大多数细胞类型的几乎完整的全基因组分类。用于测量/控制已鉴定神经元中基因表达的读出(s)是：具有归一化和绝对定量的目标基因表达水平的scRNA-seq数据和q-RT-PCR。控制组是靶基因不活跃或沉默的神经元。首先，产生了多种细胞粘附分子和其他表面大分子结构的独特资源，这些结构对设计和表征细胞特异性探针至关重要。然后，使用化学进化工具，测试了一种高通量系统，用于大规模制造细胞特异性适配体/分子信标荧光探针。最后，杂交纳米探针（例如，通过将细胞特异性荧光标记与核酸类似物偶联制成）被测试其自我递送分子结构到靶细胞的能力，而无需直接注射、电穿孔或制造转基因动物。本项目由化学学部生命过程化学项目共同资助。

项目成果

期刊论文数量（30）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Comparative neuroanatomy of ctenophores: Neural and muscular systems in Euplokamis dunlapae and related species

DOI：
10.1002/cne.24770
发表时间：
2019-10-04
期刊：
JOURNAL OF COMPARATIVE NEUROLOGY
影响因子：
2.5
作者：
Norekian, Tigran P.;Moroz, Leonid L.
通讯作者：
Moroz, Leonid L.

Life Strategies in Placozoa

Placozoa 的生活策略

DOI：
10.1101/2021.11.26.470175
发表时间：
2021
期刊：
bioRxiv
影响因子：
0
作者：
Romanova, D.Y.;Mikhail A. Nikitin, M.A.;Sergey V. Shchenkov, S.V.;Moroz, L.L.
通讯作者：
Moroz, L.L.

Glycine as a signaling molecule and chemoattractant in Trichoplax (Placozoa): insights into the early evolution of neurotransmitters

甘氨酸作为毛盘菌（Placozoa）中的信号分子和化学引诱剂：深入了解神经递质的早期进化

DOI：
10.1097/wnr.0000000000001436
发表时间：
2020
期刊：
NeuroReport
影响因子：
1.7
作者：
Romanova, Daria Y.;Heyland, Andreas;Sohn, Dosung;Kohn, Andrea B.;Fasshauer, Dirk;Varoqueaux, Frederique;Moroz, Leonid L.
通讯作者：
Moroz, Leonid L.

Development of the nervous system in the early hatching larvae of the ctenophore Mnemiopsis leidyi