| Live-cell Imaging and Quantitative Analysis of Meiotic Divisions in Caenorhabditis elegans Males
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Author:
Date:
2020-10-20
[Abstract] Live-imaging of meiotic cell division has been performed in extracted spermatocytes of a number of species using phase-contrast microscopy. For the nematode Caenorhabditis elegans, removal of spermatocytes from gonads has damaging effects, as most of the extracted spermatocytes show a high variability in the timing of meiotic divisions or simply arrest during the experiment. Therefore, we developed a live-cell imaging approach for in situ filming of spermatocyte meiosis in whole immobilized C. elegans males, thus allowing an observation of male germ cells within an unperturbed environment. For this, we make use of strains with fluorescently labeled chromosomes and centrosomes. Here we describe how to immobilize male worms for live-imaging. Further, we describe ...
[摘要] [摘要] 用相差显微镜对一些物种的精母细胞进行了减数分裂的实时成像。对于秀丽隐杆线虫来说，去除生殖腺中的精母细胞具有破坏性作用，因为大多数精母细胞在减数分裂的时间上表现出高度的变异性，或者只是在实验中停止。因此，我们开发了一种活体细胞成像方法，用于原位拍摄固定化线虫雄性精母细胞减数分裂过程，从而可以在不受干扰的环境中观察雄性生殖细胞。为此，我们利用带有荧光标记染色体和中心体的菌株。在这里我们描述如何固定男性蠕虫进行实时成像。此外，我们描述了获取和处理数据的工作流程，以获得有关精母细胞减数分裂I和II中染色体分离动态的定量信息。此外，我们最新开发的方法允许我们在电子显微镜中重新定位胶片上的主轴，而不管蜗杆的初始3D方位如何，并以统计稳健的方式分析活蠕虫的纺锤动力学。我们的实时成像方法也适用于秀丽隐杆线虫雌雄同体，并且可以扩展到其他荧光标记的线虫或其他完全透明的小型模型生物。
[背景] ...
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| Staining of Callose Depositions in Root and Leaf Tissues
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Author:
Date:
2015-03-20
[Abstract] The plant cell wall is a physical barrier, which fulfills a plethora of functions, for example it can efficiently prevent pathogen’s entry into the cell. In addition, its changing composition contributes to plants inducible defense mechanisms. This layer of defense includes pathogen perception and is followed by the activation of defense responses resulting, among others, in a modification and remodeling of the cell wall. This relatively late defense response (hours or days after contact with pathogen) comprises the accumulation of polysaccharides, such as the 1,3-ß-glucan callose, phenolic compounds and reactive oxygen species. Callose depositions occur during normal plant growth (e.g. in the phloem), they can be also a response to different stress stimuli. During the response ...
[摘要] 植物细胞壁是物理屏障，其实现过多的功能，例如它可以有效地防止病原体进入细胞。此外，其变化的组成有助于植物诱导防御机制。该防御层包括病原体感知，并且随后是防御反应的激活，导致尤其是细胞壁的修饰和重塑。这种相对较晚的防御反应（与病原体接触后的几小时或几天）包括多糖例如1,3-β-葡聚糖胼cal质，酚类化合物和活性氧簇的积累。 Callose沉积在正常植物生长期间（例如在韧皮部中）发生，它们也可以是对不同应激刺激的反应。在对病原体攻击的反应期间，胼lose质沉积是细胞壁增强的必要部分，并且对于成功的植物防御是重要的。在这里，我们描述了染色胼lose体位置斑点的方法，其可以用于量化该防御反应。
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| Stable Transformation of Cyanobacterium Synechocystis sp.
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Author:
Date:
2014-11-05
[Abstract] Cyanobacteria are prokaryotes, which perform oxygenic photosynthesis. Among them, the unicellular cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis) is a well characterized model system for studies on oxygenic photosynthesis, light signal transduction etc. Moreover, Synechocystis is applied in biotechnological applications (Desai and Atsumi, 2013). Stable transformation of Synechocytis is achieved via the uptake of DNA and incorporation into the host genome by homologous double recombination. This allows for the generation of gene knock-outs (KO) by replacing the coding sequence of the gene of interest by a KO-cassette (comprising of a selection marker flaked by sequences of the gene of interest) or stable overexpression of ...
[摘要] 蓝细菌是原核生物，其执行含氧光合作用。其中，单细胞蓝细菌集胞藻 PCC 6803（下文称为"集胞藻"）是用于研究含氧光合作用，光信号转导等的良好表征的模型系统。此外，集胞藻应用于生物技术应用（Desai和Atsumi，2013）。通过DNA的吸收和通过同源双重重组并入宿主基因组中实现集胞藻的稳定转化。这允许通过用KO盒（包含由感兴趣的基因的序列片段化的选择标记）或感兴趣的基因的某些基因的稳定过表达来产生基因敲除（KO）在相应的过表达盒插入宿主基因组上的中性插入位点之后。稳定转化集胞藻由Grigorieva和Shestakov（1982）报道。从那时起，初始协议的变体已经成功应用于变换集胞藻。在这里，我们描述了成功应用于集胞藻的稳定转化的实验室方案（Schwarzkopf等人，2014）。
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