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HEK293 cells

HEK-293

Company: ATCC
Catalog#: CRL-1573
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Analyzing (Re)Capping of mRNA Using Transcript Specific 5' End Sequencing
Author:
Date:
2020-10-20
[Abstract]  The 5′ cap is a ubiquitous feature of eukaryotic mRNAs. It is added in the nucleus onto newly synthesized pre-mRNA, and in the cytoplasm onto mRNAs after decapping or endonuclease cleavage. Cytoplasmic recapping can occur after loss of the cap at the native 5′ end, or downstream within the body of the mRNA. The identification and location of recapping events is key to understanding the functional consequences of this process. Here we present an approach that addresses this problem, using the Lexogen TeloPrime® cDNA synthesis kit to tag recapped 5′ ends. TeloPrime uses a proprietary DNA ligase to add a double stranded DNA oligonucleotide onto the 3′ end of cDNA while it is base paired with mRNA. Specificity for capped ends is obtained by the oligonucleotide having an unpaired C ... [摘要]  [摘要]5′cap是真核mRNAs普遍存在的特征。它被添加到新合成的pre-mRNA的细胞核中,并在去盖或核酸内切酶切割后加入到mRNAs的细胞质中。细胞质重拾可发生在5′端或mRNA体下游的cap缺失后。识别和定位重述事件是理解该过程的功能后果的关键。这里我们提出了一种解决这个问题的方法,使用Lexogen TeloPrime®cDNA合成试剂盒来标记重述的5′端。TeloPrime使用一种专有的DNA连接酶,在cDNA的3′端加入一个双链DNA寡核苷酸,同时与mRNA碱基配对。寡核苷酸在mRNA 5′端有一个与m7G弱碱基配对的不成对C残基。然后用引物对附加的寡核苷酸和感兴趣的mRNA进行双链cDNA的PCR扩增。得到的产物是凝胶纯化和直接测序(如果是单个带)或克隆和测序。连接的寡核苷酸和靶mRNA连接处的序列提供了cap在相应转录物上的位置。此方法适用于所有封顶转录本。它可以与Sanger测序一起用于少量的转录物,也可以用于Illumina库测序。

[背景]N7-甲基鸟苷帽是所有真核mRNAs的一个显著特征。与cap结合的蛋白质在mRNA生命周期的各个阶段发挥作用,包括核加工、输出、翻译和mRNA衰变。5′cap以共转录方式添加到所有mRNAs中,并开发了许多全基因组技术(例如,基因表达的Capped分析,或CAGE)(Morioka等人,2020年),这些技术利用末端末端的鉴定来标记转录起始位点。除了标记转录起始位点外,约25%的笼状标签映射到剪接内含子的下游(Djebali等人,2012年)。生物化学基础的证据来自我们实验室2009年的鉴定:一种细胞质复合体能够将N7甲基鸟苷帽恢复到5′-单磷酸末端的转录物上,但不能恢复到5′-羟基末端的转录物上(Otsuka等人,2009年)。细胞质封顶由一种复合酶催化,包括封盖酶(RNGTT)、帽甲基转移酶(RNMT)及其激活亚单位(RAM),以及一种将去盖转录物的5′-单磷酸末端转化为5′-二磷酸底物以进行GMP添加的激酶(Trotman和Schoenberg,2019)。我们的早期工作是基于在GMP添加步骤中阻断细胞质封盖的显性负型封盖酶的使用。该蛋白的表达导致出现许多未封顶的转录本,其末端使用5'-种族定位到下游笼状标签附近(Kiss等人,2015年;Berger等人,2019年)。虽然令人鼓舞,但这种方法有三个主要缺点:a)它需要分离带帽和未封顶的rna,b)它假设未封顶的转录本保持足够稳定,可以被检测到,以及c)它假设以这种方式检测到的未封顶末端经历了有限的额外的核外溶核修剪。 ...

Quantification of the Surface Expression of G Protein-coupled Receptors Using Intact Live-cell Radioligand Binding Assays
Author:
Date:
2020-09-20
[Abstract]  G protein-coupled receptors (GPCRs) are the most structurally diverse family of signaling proteins and regulate a variety of cell function. For most GPCRs, the cell surface is their functional destination where they are able to respond a wide range of extracellular stimuli, leading to the activation of intracellular signal transduction cascades. Thus, the quantity of receptor expression at the cell surface is a crucial factor regulating the functionality of the receptors. Over the past decades, many methods have been developed to measure the cell surface expression of GPCRs. Here, we describe an intact live-cell radioligand binding assay to quantify the surface expression of GPCRs at the endogenous levels or after overexpression. In this assay, cell cultures will be incubated with ... [摘要]  [摘要] G蛋白偶联受体(GPCR)是信号蛋白中结构最多样化的家族,可调节多种细胞功能。对于大多数GPCR,细胞表面是它们的功能目的地,能够响应广泛的细胞外刺激,从而激活细胞内信号转导级联反应。因此,受体在细胞表面的表达量是调节受体功能的关键因素。在过去的几十年中,已开发出许多方法来测量GPCR的细胞表面表达。在这里,我们描述了完整的活细胞放射性配体结合测定法,以量化内源水平或过表达后GPCR的表面表达。在该测定中,将细胞培养物与特定的细胞不可渗透的放射性配体温育,所述放射性不可配体选择性地和化学计量地结合至各个GPCR,并且通过受体结合的配体的放射性来定量细胞表面的受体数量。 此方法对于测量完整活细胞表面的功能性GPCR具有高度特异性,对于内源性,低丰度的GPCR特别有用。

[背景 ] G蛋白偶联受体(GPCR)构成细胞表面受体的最大超家族,并在生理和病理条件下调节多种细胞功能(Hauser 等人,2017; Hilger 等人,2018; Weinberg和Puthenveedu,2019) ...

Assessing Gαq/15-signaling with IP-One: Single Plate Transfection and Assay Protocol for Cell-Based High-Throughput Assay
Author:
Date:
2020-08-20
[Abstract]  Cell-based functional assays are an important part of compound screening and drug lead optimization, and they can also play a crucial role in the determination of the residues involved in ligand binding and signaling for a particular G-protein-coupled receptor. Conventional methods used for Gαq/15-coupled receptors rely on the use of fluorescent probes for Ca++ sensing (such as Fura-2 and Fluo-4) or on the incorporation of [3H]-inositol into inositol 1,4,5- triphosphate (IP3). However, these methods are not suitable for screening large libraries of compounds or for screening several mutants of the same receptor. In contrast, the IP-One assay by Cisbio is a TR-FRET assay suitable for large compound library screening when using stable cell lines that express ... [摘要]  [摘要 ] 基于细胞的功能测定法是化合物筛选和药物先导物优化的重要组成部分,并且它们可以也参与配体结合和信令残留量的测定起到至关重要的作用为一个特定的G蛋白偶联受体。用于Gα常规方法的q / 15 -偶联受体依赖于使用用于钙荧光探针++ 感测(如的Fura-2和的Fluo-4)或上掺入[的3 H] - 肌醇成肌醇1,4- ,5-三磷酸(IP3)。然而,这些方法不适合用于筛选大文库的化合物或用于筛选相同的受体的几个突变体。相反,IP-一个测定由Cisbio公司是TR-FRET测定适合大型化合物文库使用的稳定细胞株的筛选时,表达一个特定7TMR 。但是,当使用瞬时转染的7TMR突变体时,此检测方法并不理想,因为它需要两步操作进行细胞培养。因此,我们已经优化了IP-One的测定使用协议的在384孔反向转染方法的板。这为先前用于筛选Gαq / 15 偶联7TMR 的几种突变体的两步法提供了一种省时和省资源的替代方案。

[背景 ] 七跨膜受体(7TMR),也被称为G蛋白偶联受体,是超家族参与信号转导的最重要的跨膜蛋白。它们是临床批准药物的30%至50%的目标(Overington 等,2006)。Gαq / 15 偶联的7TMR激活磷脂酶Cβ(PLCβ),并产生D-肌醇1,4,5-三磷酸(IP3)和二酰基甘油(DAG)。IP3触发细胞内Ca ++ ...

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