| In vitro Engineered DNA-binding Molecule-mediated Chromatin Immunoprecipitation (in vitro enChIP) Using CRISPR Ribonucleoproteins in Combination with Next-generation Sequencing (in vitro enChIP-Seq) for the Identification of Chromosomal Interactions
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Author:
Date:
2017-11-20
[Abstract] We have developed locus-specific chromatin immunoprecipitation (locus-specific ChIP) technologies consisting of insertional ChIP (iChIP) and engineered DNA-binding molecule-mediated ChIP (enChIP). Locus-specific ChIP is a method to isolate a genomic region of interest from cells while it also identifies what binds to this region using mass spectrometry (for protein) or next generation sequencing (for RNA or DNA) as described in Fujita et al. (2016a). Recently, we identified genomic regions that physically interact with a locus using an updated form of enChIP, in vitro enChIP, in combination with NGS (in vitro enChIP-Seq) (Fujita et al., 2017a). Here, we describe a protocol on in vitro enChIP to isolate a target locus for identification of ...
[摘要] 我们开发了基因座特异性染色质免疫沉淀（基因座特异性芯片）技术，包括插入ChIP（iChIP）和工程DNA-结合分子介导ChIP（enChIP）。 基因座特异性ChIP是一种从细胞中分离感兴趣的基因组区域的方法，同时它还使用质谱（用于蛋白质）或下一代测序（用于RNA或DNA）鉴定什么与该区域结合，如Fujita等人 （2016a）。 最近，我们使用更新后的enChIP形式，结合NGS（ in vitro enChIP-Seq），鉴定了与基因座物理相互作用的基因组区域（Fujimita et al。，2017a）。 在这里，我们描述了一个体外试验的方法，用于分离靶基因座以鉴定与基因座物理相互作用的基因组区域。
【背景】阐明基因组功能强调的分子机制需要鉴定与感兴趣的基因组区域相互作用的分子。为此，我们开发了由插入ChIP（iChIP）和工程化DNA结合分子介导的ChIP（enChIP）组成的基因座特异性染色质免疫沉淀技术（基因座特异性ChIP）技术（Fujita等人 ...
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| Identification of Factors in Regulating a Protein Ubiquitination by Immunoprecipitation: a Case Study of TRF2 on Human REST4 Ubiquitination
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Author:
Date:
2015-07-05
[Abstract] Ubiquitination is the first step of the ubiquitin-proteasome pathway that regulates cells for their homeostatic functions and is an enzymatic, protein post-translational modification process in which ubiquitin is transferred to a target protein substrate by a set of three ubiquitin enzymes (Weissman et al., 2011; Bhattacharyya et al., 2014; Ristic et al., 2014). Given the importance of this process, it is plausible that ubiquitination is under strict control by many factors and that the regulatory machineries are protein-specific. An assay for the detection of a specific protein ubiquitination will enable us to examine whether a factor has a function to regulate the ubiquitination of this protein. Here we describe a protocol that detects the ubiquitination ...
[摘要] 泛素化是泛素 - 蛋白酶体通路的第一步，其调节细胞的内环境稳定功能，并且是酶，蛋白质翻译后修饰过程，其中通过一组三种泛素酶将泛素转移到靶蛋白质底物（Weissman et al。，2011; Bhattacharyya et al。，2014; Ristic 等人，2014）。考虑到这一过程的重要性，似乎可能的是，泛素化受到许多因素的严格控制，并且调节机制是蛋白质特异性的。用于检测特异性蛋白泛素化的测定将使我们能够检查因子是否具有调节该蛋白的泛素化的功能。在这里我们描述一个协议，检测人类REST4蛋白在培养细胞中的神经选择性剪接异构体（RE-1沉默转录因子），拮抗REST对神经分化和神经元形成的镇压功能的泛素化状态。使用这个协议，我们显示端粒结合蛋白TRF2通过抑制其泛素化稳定人类REST4的表达。这表明TRF2在神经分化中发挥阳性作用（Ovando-Roche等人，2014）。该方案还可用于检测其他感兴趣的蛋白质的泛素化。
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| Hepatitis C virus Cell-to-cell Spread Assay
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Author:
Date:
2014-12-20
[Abstract] Hepatitis C virus (HCV) can infect naïve cells via entry of “cell-free” extracellular virus or direct “cell-to-cell” transmission. Here, we describe an assay for detecting HCV cell-to-cell transmission, using a non-growing cell culture system that avoids confounding effects of cell growth. The assay consists of infecting a small number of cells in a confluent monolayer and then blocking subsequent cell-free extracellular virions with a neutralizing antibody such that only cell-to-cell transmission may occur. Under these conditions, incubation at 37 °C results in the formation of infected cell foci. The extent of cell-to-cell spread can then be determined by counting the number of cells in each focus. The assay may be modified to assess the effects of inhibitors and/or specific cellular ...
[摘要] 丙型肝炎病毒（HCV）可通过"无细胞"细胞外病毒的进入或直接的"细胞到细胞"传递而感染初始细胞。 在这里，我们描述检测HCV细胞到细胞传播，使用一个非增长的细胞培养系统，避免细胞生长的混杂效应的测定。 该测定法包括感染汇合单层中的少量细胞，然后用中和抗体阻断随后的无细胞的细胞外病毒粒子，使得仅可发生细胞与细胞的传递。 在这些条件下，在37℃下孵育导致感染的细胞病灶的形成。 然后可以通过计数每个焦点中的细胞数量来确定细胞与细胞扩散的程度。 可以修改测定以评估抑制剂和/或特异性细胞基因对HCV的细胞与细胞扩散的影响。
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