| A Method for Extracting the Nuclear Scaffold from the Chromatin Network
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Author:
Date:
2018-04-20
[Abstract] Each cell contains many large DNA polymers packed in a nucleus of approx. 10 μm in diameter. With histones, these DNA polymers are known to form chromatins. How chromatins further compact in the nucleus is unclear but it inevitably depends on an extensive non-chromatin nuclear scaffold. Imaging of endogenous chromatin network and the complementary scaffold that support this network has not been achieved but biochemical and proteomic investigations of the scaffold can still provide important insights into this chromatin-organizing network. However, this demands highly inclusive and reproducible extraction of the nuclear scaffold. We have recently developed a simple protocol for releasing the scaffold components from chromatins. The inclusiveness of the extract was testified by the ...
[摘要] 每个细胞都含有许多大型DNA聚合物，其中包含大约一个核。直径10微米。用组蛋白，已知这些DNA聚合物形成染色质。染色质在核中如何进一步致密还不清楚，但它不可避免地依赖于广泛的非染色质核支架。内源性染色质网络的成像和支持该网络的互补支架尚未实现，但支架的生化和蛋白质组学研究仍然可以提供关于该染色质组织网络的重要见解。但是，这需要高度包容和可重复的提取核支架。我们最近开发了一个简单的协议，用于从染色质中释放脚手架组件。提取物的包容性由以下观察结果证实：当从核中提取时，剩余的核染色质被释放为延伸且通常平行的染色质纤维。基本上，该方案包括纯核的产生，用Triton X-100处理细胞核以产生包膜消耗的细胞核（TxN），并在含蔗糖的缓冲液中在500mM NaCl中提取细胞核。 TxN的这个组合提取被称为TxNE。
【背景】通过蛋白质和核糖核蛋白的复杂支架，染色质在细胞核中密集并动态地压缩。与细胞骨架网络不同（Fischer和Fowler，2015），对这种核支架的显微观察在技术上是具有挑战性的。这可能反映了每个细胞核内染色质的主导地位，支架与细胞核交织在一起。核的球形排列也对成像这种支架结构造成挑战。核支架的主要元素是核层（NL）（Gruenbaum和Foisner，2015）。 ...
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| Genome-Wide siRNA Screen for Anti-Cancer Drug Resistance in Adherent Cell Lines
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Author:
Date:
2015-05-20
[Abstract] The expression of genes is frequently manipulated in cell lines to study their cellular functions. The use of exogenous small Interfering RNAs (siRNAs) is a very efficient technique to temporarily downregulate the expression of genes of interest [reviewed by Hannon and Rossi (2004)]. A genome-wide siRNA library allows the user to study both the effect of each individual gene on a particular cell phenotype in a high throughput manner and also assess its phenotypic effect relative to all other genes targeted. Several factors that potentially influence the outcome of a screen need to be considered when performing a large siRNA screen (Jiang et al., 2011). Here we present a detailed protocol for a genome-wide screen to identify genes involved in anti-cancer drug resistance using the ...
[摘要] 基因的表达经常在细胞系中操作以研究它们的细胞功能。外源小干扰RNA（siRNA）的使用是临时下调感兴趣的基因的表达的非常有效的技术[由Hannon和Rossi（2004）综述]。全基因组siRNA文库允许用户以高通量方式研究每个个体基因对特定细胞表型的作用，并且还评估其相对于所有其他靶向基因的表型效应。当进行大siRNA筛选时，需要考虑潜在地影响筛选结果的几个因素（Jiang等人，2011）。在这里，我们提出了一个详细的协议，使用人类siGENOME库从Dharmacon的基因组范围屏幕识别参与抗癌耐药性的基因。在该方案中，我们关注对表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKI）厄洛替尼在对EGFR-TKIs敏感的肺癌细胞系PC9中的治疗的抗性（de Bruin等。，2014）。该方案可用于其他细胞系和其他药物治疗，我们在下面的注释中扩展。
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