Fluorescence stereomicroscope imaging systems

荧光体视显微镜用于斑马鱼胚胎和幼体的活体成像，彻底改变了我们的 能够深入了解人类发育和疾病过程。它允许真实的时间可视化 构成脊椎动物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，导致细胞机制的新发现 可以通过操纵来改善人类健康。大量的单细胞表达数据 从不同的发育阶段和组织产生的，提供了丰富的新信息， 决定细胞谱系的关键转录因子。到目前为止，创建转录因子特异性 细胞谱系追踪报告基因，如Cre重组酶，效率低下，并且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精准靶向整合策略， 可以在任何感兴趣的基因中产生Cre驱动程序，从而释放出检查细胞谱系的潜力， 以前无法获得的，并将单细胞基因表达数据转化为细胞功能工具， 谱系特异性分析。随着我们的R24资源赠款，我们已经恢复Cre驱动程序的转录 因子基因定义中胚层和免疫细胞谱系，并将其扩展到早期 中内胚层、血管和造血细胞。荧光显微镜是 我们正在建设的转基因资源和功能性荧光立体显微镜设备对于 高效地生成和表征新的线。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的能力。在目标1中，我们建议提供资金， 通过购买3台Leica M165 FC成像系统来支持我们的成像能力， 每日筛选和记录新的转基因品系。在目标2中，我们建议提供资金， 通过购买1台Leica M205 FCA，可实现细胞谱系标记的真实的实时成像 雷电成像系统。总之，这些系统将使我们能够成功地培养斑马鱼Cre 驱动器社区资源，并彻底表征空间和时间细胞谱系标记 每个Cre线的活动。

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.