| A Pulse–chase EdU Method for Detection of Cell Division Orientation in Arabidopsis and Juncus prismatocarpus Leaf Primordia
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Author:
Date:
2021-01-05
[Abstract] In plants, the morphological diversity of leaves is largely determined by cell division, especially cell division orientation. Whereas cell division itself is easily monitored, the detection and quantification of cell division orientation are difficult. The few existing methods for detection and quantification of cell division orientation are either inefficient or laborious. Here, we describe a pulse-chase strategy using a 5-ethynyl-2’-deoxyuridine (EdU) labeling assay. Plant tissues are first incubated with EdU for a short period (pulse), followed by a long incubation without EdU (chase). Using this method, the positions of daughter cells are easily detected and can be used to quantify cell division orientation. Our protocol is rapid and very efficient for quantitative analysis of ...
[摘要] [摘要]在植物中，叶片的形态多样性在很大程度上取决于细胞分裂，尤其是细胞分裂方向。尽管细胞分裂本身很容易监测，但是细胞分裂方向的检测和定量却很困难。现有的几种检测和定量细胞分裂方向的方法要么效率低下要么费力。在这里，我们描述了使用5-乙炔基-2'-脱氧尿苷（EdU ）标记测定的脉冲追踪策略。首先将植物组织与EdU一起短时间（脉冲）孵育，然后在没有EdU的情况下长时间孵育（追逐）。使用这种方法，子细胞的位置易于检测，可用于量化细胞分裂方向。我们的协议可以快速有效地定量分析细胞分裂方向，并且可以同时应用于模型植物和非模型植物。
图形摘要：
通过脉冲追逐EdU方法清晰可见的植物细胞分裂对
[背景]植物细胞通过细胞壁彼此附接，并且不能迁移。因此，在叶片发育的早期，组织化，定向的细胞分裂在很大程度上决定了成熟叶片的形状。迄今为止，还没有报道用于有效和快速检测和定量细胞分裂取向的方法。现有方法包括使用ap CYCB1; 1 :: GUS （β-葡萄糖醛酸糖苷酶）报告基因线（末期）可视化子核（末期）（Horiguchi et al。，2011）或使用4'，6-diamidino可视化纺锤状赤道（中期） -2-苯基吲哚（DAPI）染色（Fukushima et ...
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| Estimation of the Minimum Number of Replication Origins Per Chromosome in any Organism
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Author:
Date:
2020-10-20
[Abstract] Eukaryote nuclear genomes predominantly replicate through multiple replication origins. The number of replication origins activated per chromosome during the S-phase duration may vary according to many factors, but the predominant one is replication stress. Several studies have applied different approaches to estimate the number and map the positions of the replication origins in various organisms. However, without a parameter to restrict the minimum of necessary origins, less sensitive techniques may suggest conflicting results. The estimation of the minimum number of replication origins (MO) per chromosome is an innovative method that allows the establishment of a threshold, which serves as a parameter for genomic approaches that map origins. For this, the MO can be easily ...
[摘要] [摘要] 比率可能因多种因素而变化，但最主要的因素是复制应力。一些研究应用了不同的方法来估计复制源在不同生物体中的数量和位置。然而，如果没有一个参数来限制必要起源的最小值，那么不太敏感的技术可能会产生相互矛盾的结果。估计每个染色体的最小复制源数量（MO）是一种创新的方法，它允许建立一个阈值，作为绘制起源的基因组方法的参数。为此，MO可以很容易地通过一个公式得到，这个公式需要作为参数：染色体大小、S期持续时间和复制率。在基因组数据库（如NCBI）中可以获得任何生物体的染色体大小，通过监测DNA复制来估计S期的持续时间，并通过DNA组合方法获得复制率。 提供了一种简单、快速的估算MO的方法一个新的方法学框架来协助研究任何有机体。
关键词： DNA复制，复制来源，复制率，S期持续时间，染色体大小
[背景] ...
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| Whole-mount Enteroid Proliferation Staining
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Author:
Date:
2016-06-20
[Abstract] Small intestinal organoids, otherwise known as enteroids, have become an increasingly utilized model for intestinal biology in vitro as they recapitulate the various epithelial cells within the intestinal crypt (Mahe et al., 2013; Sato et al., 2009). Assessment of growth dynamics within these cultures is an important step to understanding how alterations in gene expression, treatment with protective and toxic agents, and genetic mutations alter properties essential for crypt growth and survival as well as the stem cell properties of the individual cells within the crypt. This protocol describes a method of visualization of proliferating cells within the crypt in three dimensions (Barrett et al., 2015). Whole-mount proliferation staining of enteroids ...
[摘要] 小肠类器官，也称为肠袢，已经成为越来越多地用于肠道生物学的体外模型，因为它们重现了在肠隐窝内的各种上皮细胞（Mahe等人 ，2013; Sato et al。，2009）。在这些培养物中评估生长动力学是理解基因表达的改变，用保护性和毒性试剂处理以及遗传突变改变隐窝生长和存活所必需的性质以及细胞内单个细胞的干细胞性质的重要步骤地穴。该方案描述了在隐窝内三维的增殖细胞的可视化方法（Barrett等人，2015）。使用EdU掺入的肠衣的整体增殖染色使得研究者能够观察到肠内的所有增殖细胞，而不是如在包埋和切片中所见到的在薄切片中获得生长信息，确保来自干细胞区室的增殖的真实表现到隐窝的终末分化细胞。
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