Fluorescence stereomicroscope imaging systems

用荧光立体显微镜对斑马鱼胚胎和幼虫进行实时成像，使我们的 洞察人类发育和疾病过程的能力。它允许实时可视化 构成脊椎动物的复杂组织和器官的细胞谱系。研究如何 这些过程被损伤或疾病破坏，导致对细胞机制的新发现 这可以被操纵来改善人类健康。海量的单细胞表达数据 从不同的发育阶段和组织中产生的，提供了关于 指定细胞谱系的关键转录因子。到目前为止，创造转录因子特异性的方法 细胞谱系追踪报告，如Cre重组酶，一直效率低下，并受到以下能力的限制 克隆基因特异性启动子。通过我们的GeneWeld CRISPR精确定向集成战略，我们 可以在任何感兴趣的基因中生成Cre驱动程序，从而解锁了检查 以前无法访问，并将单细胞基因表达数据转换为细胞的功能工具 特定血统的分析。使用我们的R24资源拨款，我们已经恢复了转录中的CRE驱动程序 定义中胚层和免疫细胞谱系的因子基因，并将其扩展到早期 中胚层、血管和造血细胞。荧光显微镜是科学研究的核心 我们正在建设的转基因资源和功能荧光立体显微镜设备对 高效地生成和表征新的线条。为了满足R24的成像需求，我们提出了两个目标 这将大大扩展我们的荧光立体显微镜能力。在目标1中，我们建议提供资金以 通过购买3个徕卡M165 FC成像系统来支持我们的成像能力 新转基因品系的日常筛选和记录。在目标2中，我们建议提供资金来提升我们的 通过购买1个徕卡M205 FCA进行细胞谱系标记的实时实时成像能力 雷电成像系统。这些系统结合在一起，将使我们能够成功地种植斑马鱼CRE 驱动社区资源并彻底表征空间和时间细胞谱系标记 每条CRE行的活动。

Fluorescence stereomicroscopy for live imaging of zebrafish embryos and larvae has revolutionized our ability to gain insight into human developmental and disease processes. It allows visualization in real time of cell lineages that build the complex tissues and organs that define vertebrate animals. Examining how these processes are disrupted by injury or disease leads to novel discoveries of the cellular mechanisms that can be manipulated to improve human health. The vast amount of single cell expression data generated from distinct developmental stages and tissues has provided a wealth of new information about key transcription factors that specify cell lineages. Until now, methods to create transcription factor-specific cell lineage tracing reporters, such as Cre recombinase, have been inefficient and limited by the ability to clone gene specific promoters. With our GeneWeld CRISPR precision targeted integration strategy, we can generate a Cre driver in any gene of interest, unlocking the potential to examine cell lineages that were previously inaccessible, and to transform single cell gene expression data into functional tools for cell lineage specific analysis. With our R24 resource grant, we have recovered Cre drivers in transcription factor genes that define mesodermal and immune cell lineages and are expanding this to early mesendoderm, vascular, and hematopoietic cells. Fluorescence microscopy is at the heart of the transgenic resource we are building and functional fluorescence stereomicroscope equipment is critical to efficiently generate and characterize new lines. To meet the imaging needs of the R24, we propose 2 aims that will significantly expand our fluorescence stereomicroscope capabilities. In Aim 1 we propose funds to support our imaging capabilities through the purchase of 3 Leica M165 FC imaging systems for routine daily screening and documentation of new transgenic lines. In Aim 2 we propose funds to elevate our capacity for real time live imaging of cell lineage labeling through the purchase of 1 Leica M205 FCA Thunder imaging system. Together, these systems will allow us to successfully grow the zebrafish Cre driver community resource and thoroughly characterize the spatial and temporal cell lineage labeling activity of each Cre line.