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Thermo ScientificTM NuncTM 15mL & 50mL Conical Sterile Polypropylene Centrifuge Tubes

Nunc TM 15mL&50mL圆锥形无菌聚丙烯离心管

Company: Thermo Fisher Scientific
Catalog#: 339651
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Sleeping Beauty Transposon-based System for Rapid Generation of HBV-replicating Stable Cell Lines
Author:
Date:
2018-07-05
[Abstract]  The stable HBV-transfected cell lines, which based on stable integration of replication-competent HBV genome into hepatic cells, are widely used in basic research and antiviral drug evaluation against HBV. However, previous reported strategies to generate HBV-replicating cell lines, which primarily rely on random integration of exogenous DNA by plasmid transfection, are inefficient and time-consuming. We newly developed an all-in-one Sleeping Beauty transposon system (denoted pTSMP-HBV vector) for robust generation of stable HBV-replicating cell lines of different genotype. The pTSMP-HBV vector contains HBV 1.3-copy genome and dual selection markers (mCherry and puromycin resistance gene), allowing rapid enrichment of stably-transfected cells via red fluorescence-activated cell sorting ... [摘要]  稳定的HBV转染细胞系基于将复制能力的HBV基因组稳定整合到肝细胞中,广泛用于基础研究和针对HBV的抗病毒药物评估。然而,先前报道的产生HBV复制细胞系的策略(其主要依赖于通过质粒转染的外源DNA的随机整合)是低效且耗时的。我们新开发了一体化睡眠美容转座子系统(表示为pTSMP-HBV载体),用于稳定产生不同基因型的稳定HBV复制细胞系。 pTSMP-HBV载体含有HBV1.3拷贝基因组和双重选择标记(mCherry和嘌呤霉素抗性基因),允许通过红色荧光激活细胞分选和嘌呤霉素抗生素选择快速富集稳定转染的细胞。在该方案中,我们描述了构建HBV复制稳定细胞和系统评估这些细胞的HBV复制和病毒蛋白表达谱的详细程序。

【背景】慢性乙型肝炎病毒(HBV)感染目前是一个主要的公共卫生负担,影响全球超过2.4亿人(Witt-Kehati et al。,2016)。慢性HBV患者患慢性活动性肝炎,肝硬化或原发性肝细胞癌(HCC)的风险升高(Schweitzer et al。,2015)。目前用干扰素-α或核苷类似物治疗并不能根除病毒,它们对清除乙型肝炎表面抗原(HBsAg)的作用有限(Lucifora和Protzer,2016; Soriano et al。,2017) 。因此,迫切需要开发新的抗病毒抑制剂(Nassal,2015)。

用于评估新药抗HBV活性的细胞培养模型是新药开发的重要工具。稳定的HBV复制细胞系,携带复制能力的HBV基因组稳定整合到人肝癌细胞系(Huh7和/或HepG2)的基因组中,被广泛用于评估抗病毒药物的作用(Witt-Kehati ...

Nuclei Isolation from Nematode Ascaris
Author:
Date:
2017-05-05
[Abstract]  Preparing nuclei is necessary in a variety of experimental paradigms to study nuclear processes. In this protocol, we describe a method for rapid preparation of large number of relatively pure nuclei from Ascaris embryos or tissues that are ready to be used for further experiments such as chromatin isolation and ChIP-seq, nuclear RNA analyses, or preparation of nuclear extracts (Kang et al., 2016; Wang et al., 2016). [摘要]  在各种实验范例中准备核是必要的,以研究核过程。在本协议中,我们描述了一种从准备用于进一步实验(如染色质分离和ChIP-seq,核RNA)的蛔虫胚胎或组织快速制备大量相对纯的核的方法分析或准备核提取物(Kang等人,2016; Wang等人,2016)。

背景 核分离通常是研究核事件的分子和生物化学方面的第一步。已经开发了几种方法来分离来自不同组织和细胞类型的细胞核。然而,除了C以外的线虫的细胞核分离方案很少。已经描述了线索,(Ooi等人,2010; Zanin等人,2011; Haenni等人,2012, )。已经使用寄生线虫蛔虫的胚胎来制备用于体外无细胞系统的各种提取物(Cohen等人,2004) ; Lall等人,2004)。然而,这些提取物通常是全细胞提取物。在这里,我们描述了从线虫蛔虫制备细胞核的方法。

Whole-mount Enteroid Proliferation Staining
Author:
Date:
2016-06-20
[Abstract]  Small intestinal organoids, otherwise known as enteroids, have become an increasingly utilized model for intestinal biology in vitro as they recapitulate the various epithelial cells within the intestinal crypt (Mahe et al., 2013; Sato et al., 2009). Assessment of growth dynamics within these cultures is an important step to understanding how alterations in gene expression, treatment with protective and toxic agents, and genetic mutations alter properties essential for crypt growth and survival as well as the stem cell properties of the individual cells within the crypt. This protocol describes a method of visualization of proliferating cells within the crypt in three dimensions (Barrett et al., 2015). Whole-mount proliferation staining of enteroids ... [摘要]  小肠类器官,也称为肠袢,已经成为越来越多地用于肠道生物学的体外模型,因为它们重现了在肠隐窝内的各种上皮细胞(Mahe等人 ,2013; Sato et al。,2009)。在这些培养物中评估生长动力学是理解基因表达的改变,用保护性和毒性试剂处理以及遗传突变改变隐窝生长和存活所必需的性质以及细胞内单个细胞的干细胞性质的重要步骤地穴。该方案描述了在隐窝内三维的增殖细胞的可视化方法(Barrett等人,2015)。使用EdU掺入的肠衣的整体增殖染色使得研究者能够观察到肠内的所有增殖细胞,而不是如在包埋和切片中所见到的在薄切片中获得生长信息,确保来自干细胞区室的增殖的真实表现到隐窝的终末分化细胞。

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