Fluorescent Polysome Profiling in Caenorhabditis elegans
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Author:
Date:
2020-09-05
[Abstract] An important but often overlooked aspect of gene regulation occurs at the level of protein translation. Many genes are regulated not only by transcription but by their propensity to be recruited to actively translating ribosomes (polysomes). Polysome profiling allows for the separation of unbound 40S and 60S subunits, 80S monosomes, and actively translating mRNA bound by two or more ribosomes. Thus, this technique allows for actively translated mRNA to be isolated. Transcript abundance can then be compared between actively translated mRNA and all mRNA present in a sample to identify instances of post-transcriptional regulation. Additionally, polysome profiling can be used as a readout of global translation rates by quantifying the proportion of actively translating ribosomes within a ...
[摘要] [摘要 ] 基因调控的一个重要但经常被忽视的方面发生在蛋白质翻译的水平。许多基因不仅受转录调控,还受其被募集以主动翻译核糖体(多核糖体)的倾向性的调节。多核糖体分析允许分离未结合的40S和60S亚基,80S单核糖体,并主动翻译由两个或多个核糖体结合的mRNA。因此,该技术允许分离出主动翻译的mRNA。然后可以在主动翻译的mRNA和样品中存在的所有mRNA之间比较转录本丰度,以鉴定转录后调控的实例。此外,多核糖体分析可以用作ar 通过量化样品中活性翻译核糖体的比例来确定总体翻译率。先前建立的多核糖体谱分析方案依赖于RNA的吸收来可视化级分中多核糖体的存在。但是,随着能够检测荧光的流通池的出现,除了RNA的吸收以外,还可以检测和定量荧光标记的蛋白质与多核糖体的结合。该协议提供了有关如何在秀丽隐杆线虫中进行荧光多核糖体分析以收集主动翻译的mRNA,定量整体翻译的变化以及检测核糖体结合伴侣的详细说明。
[背景 ] 绑定到同一个mRNA转录多积极核糖体的翻译被称为多聚核糖体。可使用多核糖体分析将多核糖体与其他核糖体形式和未结合的mRNA分离。P olysome 分析已经在蛋白质翻译领域的关键技术。与mRNA的-SEQ的同时,多边形OME分析允许由转录后机制调节以进行检测和定量转录物(兰等人,2019;罗林。等人,2019) ,通过定量PCR (熊猫等人, ...
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In vitro Differentiation of Murine Innate Lymphoid Cells from Common Lymphoid Progenitor Cells
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Author:
Date:
2016-03-20
[Abstract] Subtypes of innate lymphoid cells (ILC), defined based on their cytokine secretion profiles and transcription factor expression, are important for host protection from pathogens and maintaining tissue homeostasis. ILCs develop from common lymphoid progenitors (CLP) in the bone marrow. Using the methods described here, we have previously shown that loss of the transcriptional regulator TOX (Thymocyte-selection associated HMG-box protein) leads to specific changes in ILC development and differentiation. Here, we describe how to obtain ILCs from in vivo isolated CLP grown in vitro.
[摘要] 先天淋巴细胞(ILC)的亚型,基于其细胞因子分泌谱和转录因子表达来定义,对于宿主保护免于病原体和维持组织内稳态是重要的。 ILC从骨髓中的普通淋巴祖细胞(CLP)发展而来。 使用本文所述的方法,我们以前显示转录调节器TOX(胸腺细胞选择相关的HMG框蛋白)的损失导致ILC发展和分化的具体变化。 在这里,我们描述了如何从体内分离的CLP获得ILC。
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