| Single-molecule Analysis of DNA Replication Dynamics in Budding Yeast and Human Cells by DNA Combing
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Author:
Date:
2017-06-05
[Abstract] The DNA combing method allows the analysis of DNA replication at the level of individual DNA molecules stretched along silane-coated glass coverslips. Before DNA extraction, ongoing DNA synthesis is labeled with halogenated analogues of thymidine. Replication tracks are visualized by immunofluorescence using specific antibodies. Unlike biochemical and NGS-based methods, DNA combing provides unique information on cell-to-cell variations in DNA replication profiles, including initiation and elongation. Finally, this assay can be used to monitor the effect of DNA lesions on fork progression, arrest and restart.
[摘要] DNA梳理方法允许在沿着硅烷涂覆的玻璃盖玻片拉伸的单个DNA分子的水平上分析DNA复制。在DNA提取前，进行的DNA合成用胸苷的卤化类似物标记。使用特异性抗体通过免疫荧光可视化复制轨迹。与生物化学和基于NGS的方法不同，DNA梳理提供了DNA复制谱中细胞间细胞变化的独特信息，包括引发和延长。最后，该测定可用于监测DNA损伤对叉进展，停止和重新启动的影响。
背景 在称为复制起点的真核染色体上的数千个位点处启动DNA合成。原始激活遵循由检查点激酶和染色质的表观遗传修饰（Prioleau和MacAlpine，2016）控制的定义良好的复制计时程序。复制叉在正常S阶段经常停顿。叉停止是由多个事件引起的，例如DNA损伤，紧密结合的蛋白质复合物和高表达基因的转录（Tourriere和Pasero 2007; Zeman and Cimprich，2013）。真核生物已经制定了不同的策略来应对这种复制压力，包括修复机制来重新启动捕获的叉子和激活休眠复制起源以抢救终末抓捕的叉。
＆NBSP; ...
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| Heterochronic Pellet Assay to Test Cell-cell Communication in the Mouse Retina
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Author:
Date:
2017-02-05
[Abstract] All seven retinal cell types that make up the mature retina are generated from a common, multipotent pool of retinal progenitor cells (RPCs) (Wallace, 2011). One way that RPCs know when sufficient numbers of particular cell-types have been generated is through negative feedback signals, which are emitted by differentiated cells and must reach threshold levels to block additional differentiation of that cell type. A key assay to assess whether negative feedback signals are emitted by differentiated cells is a heterochronic pellet assay in which early stage RPCs are dissociated and labeled with BrdU, then mixed with a 20-fold excess of dissociated differentiated cells. The combined cells are then re-aggregated and cultured as a pellet on a membrane for 7-10 days in vitro. During ...
[摘要] 构成成熟视网膜的所有七种视网膜细胞类型都是由普通的多能视网膜祖细胞池（RPC）产生的（Wallace，2011）。已经产生足够数量的特定细胞类型的RPC知道的一种方式是通过负反馈信号，其由分化细胞发射并且必须达到阈值水平以阻止该细胞类型的额外分化。评估负反馈信号是否由分化细胞发出的关键测定是异源沉淀测定，其中早期RPC被解离并用BrdU标记，然后与20倍过量的解离的分化细胞混合。然后将组合的细胞再次聚集并在细胞膜上培养7-10天。在这段时间内，RPC将会分化，BrdU + RPC的命运可以使用细胞类型特异性标记进行评估。开发这种沉淀测定的研究人员最初表明，当两种细胞类型混合在一起时，新生儿RPC与胚胎RPC相比，在加速进度条件下产生杆（Watanabe和Raff，1990; Watanabe等，1997）。我们已经使用这种测定来证明我们发现作为视网膜神经节细胞（RGC）分化的负调节物的声刺猬（Shh）促进RPC增殖（Jensen和Wallace，1997; Ringuette等，2014）。最近我们修改了异质性沉淀测定法，以评估视网膜无长突细胞的反馈信号的作用，将转化生长因子β2（Tgfβ2）鉴定为负反馈信号，并将Pten作为Tgfβ2应答的调节剂（Ma et al。，2007 ; ...
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