A Method for SUMO Modification of Proteins in vitro
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Author:
Date:
2018-10-05
[Abstract] The Small Ubiquitin-related Modifier (SUMO) is a protein that is post-translationally added to and reversibly removed from other proteins in eukaryotic cells. SUMO and enzymes of the SUMO pathway are well conserved from yeast to humans and SUMO modification regulates a variety of essential cellular processes including transcription, chromatin remodeling, DNA damage repair, and cell cycle progression. One of the challenges in studying SUMO modification in vivo is the relatively low steady-state level of a SUMO-modified protein due in part to the activity of SUMO deconjugating enzymes known as SUMO Isopeptidases or SENPs. Fortunately, the use of recombinant SUMO enzymes makes it possible to study SUMO modification in vitro. Here, we describe a sensitive method for ...
[摘要] 小泛素相关修饰物(SUMO)是一种蛋白质,其翻译后添加到真核细胞中并可逆地从其他蛋白质中去除。 SUMO和SUMO途径的酶从酵母到人类都很保守,SUMO修饰调节了多种基本细胞过程,包括转录,染色质重塑,DNA损伤修复和细胞周期进程。 研究SUMO修饰体内的挑战之一是SUMO修饰蛋白的相对低的稳态水平,部分原因是SUMO去缀合酶(SUMO Isopeptidases或SENPs)的活性。 幸运的是,使用重组SUMO酶可以在体外研究SUMO修饰。 在这里,我们描述了一种灵敏的方法,用于检测目标人类蛋白质的SUMO修饰,使用来自兔网织红细胞和放射性标记的氨基酸的体外转录和翻译系统。 【背景】与其他泛素蛋白家族修饰一样,SUMO修饰通过ATP依赖性酶促级联发生,涉及E1激活酶(人类中的Aos1 / Uba2异二聚体),E2结合酶(Ubc9)和许多E3连接之一的连续活性。酶(Gareau和Lima,2010)。具有SUMO缀合共有位点的蛋白质ΨKxE(Ψ是疏水残基,其后是赖氨酸,任何氨基酸和谷氨酸),可以通过哺乳动物中表达的一种或几种SUMO旁系同源物(包括SUMO1,SUMO2)进行有效修饰。或SUMO3(统称为SUMO2 / 3,因为它们的序列同源性为97%)(Gareau和Lima,2010; Flotho和Melchior,2013)。 ...
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Generating Loss-of-function iPSC Lines with Combined CRISPR Indel Formation and Reprogramming from Human Fibroblasts
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Author:
Date:
2018-04-05
[Abstract] For both disease and basic science research, loss-of-function (LOF) mutations are vitally important. Herein, we provide a simple stream-lined protocol for generating LOF iPSC lines that circumvents the technical challenges of traditional gene-editing and cloning of established iPSC lines by combining the introduction of the CRISPR vector concurrently with episomal reprogramming plasmids into fibroblasts. Our experiments have produced nearly even numbers of all 3 genotypes in autosomal genes. In addition, we provide a detailed approach for maintaining and genotyping 96-well plates of iPSC clones.
[摘要] 对于疾病和基础科学研究而言,功能丧失(LOF)突变是非常重要的。 在这里,我们提供了一个简单的流线化协议来产生LOF iPSC系列,通过将CRISPR载体与附加型重编程质粒同时引入成纤维细胞,规避了传统基因编辑和已建立的iPSC系的克隆的技术挑战。 我们的实验已经产生了常染色体基因中所有3种基因型的几乎偶数。 此外,我们提供了一个详细的方法来维护和iPSC克隆的96孔板的基因分型。
【背景】CRISPR / Cas9技术允许简单且特异地针对特定基因组位置进行基因编辑。将该技术与诱导性多能干细胞(iPSC)的疾病建模和再生医学潜力相结合将继续对生物医学研究产生前所未有的影响。然而,使CRISPR / Cas9系统适应iPSC已经提出了几个挑战。在细胞系中进行基因编辑的传统方法是用表达Cas9蛋白质的质粒和指导RNA(gRNA)转染细胞,然后产生单克隆并筛选所需的遗传改变。不幸的是,iPSC不适用于单细胞克隆。已经开发了几种补充媒介和克隆方法来克服这一困难,但仍然充满昂贵的设备(低氧培养箱),困难的技术步骤(FACS分选的单个iPSC的存活)或劳动密集型方案(亚克隆)(Forsyth ,2006; Miyaoka ...
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