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Thermo Fisher Scientific

Company: Thermo Fisher Scientific
Catalog#: 14040133
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Sendai Virus Propagation Using Chicken Eggs
Author:
Date:
2018-09-20
[Abstract]  Sendai virus is a member of the family Paramyxoviridae, and an enveloped virus with a negative-stranded RNA genome. Sendai virus is not pathogenic to humans, but for mice and can cause pneumonia in mice. Easy and efficient techniques for propagating Sendai virus are required for studying virus replication, virus-induced innate- and adaptive-immunity, Sendai-virus-based virotherapy and IgA nephropathy. Here, we describe a protocol for Sendai virus propagation using chicken eggs. This traditional protocol enables us to generate a large amount of virus enough for animal experiments as well as cell culture experiments in a relatively inexpensive way. [摘要]  仙台病毒是家族副粘病毒科的成员,以及具有负链RNA基因组的包膜病毒。 仙台病毒对人类无致病性,但对小鼠而言可导致小鼠肺炎。 传播仙台病毒的简便有效的技术是研究病毒复制,病毒诱导的先天免疫和适应性免疫,基于仙台病毒的病毒疗法和IgA肾病所必需的。 在这里,我们描述了使用鸡蛋传播仙台病毒的协议。 这种传统的方案使我们能够以相对便宜的方式产生足够的大量病毒用于动物实验以及细胞培养实验。

【背景】仙台病毒(SeV)是一种小鼠副流感病毒1型,于20世纪50年代在日本仙台发现(Ishida和Homma,1978)。该病毒曾被日本病毒学会命名为日本血凝病毒(HVJ),后来被称为“新生病毒性肺炎(仙台型)”(Kuroya和Ishida,1953)。 SeV这个名字目前最受欢迎,现在被认为是老鼠的病原体,而不是人类(Karron RA,2007)。 Fukumi 等人首次描述了1954年小鼠的SeV感染(Fukumi et al。,1954)。这种感染可能是亚临床的,但SeV也被认为是某些小鼠品系中肺炎的主要原因之一(Fukumi et al。,1954; Parker et al。, 1978年)。 SeV是研究以下各个领域的优秀工具:IgA肾病小鼠模型的病理机制(Yamashita et al。,2007; Chintalacharuvu et al。, ...

A Lentiviral Pseudotype ELLA for the Measurement of Antibodies Against Influenza Neuraminidase
Author:
Date:
2018-07-20
[Abstract]  This protocol describes the rapid and safe production of lentiviral pseudotypes characterized by a lentiviral core containing a reporter, in conjunction with avian influenza haemagglutinin (HA) and human neuraminidase (NA) glycoproteins on the surface. Production is optimized with Endofectin LentiTM transfection reagent in 6-well plate format. These pseudotyped viruses can be employed for serological assays of surface glycoproteins HA and NA. They can be efficiently used to perform the ELLA (Enzyme-linked lectin assay) to measure NA inhibiting antibodies in lieu of using reassortant virus or Triton X-100 inactivated wild-type virus as source of antigen, which may require higher biosafety levels. [摘要]  该方案描述了慢病毒假型的快速和安全生产,其特征在于含有报道分子的慢病毒核心,以及表面上的禽流感血凝素(HA)和人神经氨酸酶(NA)糖蛋白。 使用6孔板形式的Endofectin Lenti TM 转染试剂优化生产。 这些假型病毒可用于表面糖蛋白HA和NA的血清学测定。 它们可以有效地用于进行ELLA(酶联凝集素测定)以测量NA抑制抗体,而不是使用重配病毒或Triton X-100灭活的野生型病毒作为抗原来源,这可能需要更高的生物安全水平。

【背景】流感病毒假型的产生先前已被广泛描述(Nefkens et al。,2007; Temperton et al。,2007; Carnell et al。,2015)。需要一种安全快速的系统来评估通过ELLA测定靶向NA的抗体,避免使用重配错配病毒或野生型病毒,这已经通过产生携带NA型流感假型来满足(Prevato et al。,2015)。最近的一项研究(Biuso et al。,2017)证实HA与NA的共表达改善了新形成的假型慢病毒的释放。在这里,我们报告了一种简单,广泛适用和优化的PV生产方案,使用6孔板格式的Endofectin Lenti TM ...

Detection and Analysis of Circular RNAs by RT-PCR
Author:
Date:
2018-03-20
[Abstract]  Gene expression in eukaryotic cells is tightly regulated at the transcriptional and posttranscriptional levels. Posttranscriptional processes, including pre-mRNA splicing, mRNA export, mRNA turnover, and mRNA translation, are controlled by RNA-binding proteins (RBPs) and noncoding (nc)RNAs. The vast family of ncRNAs comprises diverse regulatory RNAs, such as microRNAs and long noncoding (lnc)RNAs, but also the poorly explored class of circular (circ)RNAs. Although first discovered more than three decades ago by electron microscopy, only the advent of high-throughput RNA-sequencing (RNA-seq) and the development of innovative bioinformatic pipelines have begun to allow the systematic identification of circRNAs (Szabo and Salzman, 2016; Panda et al., 2017b; Panda et al., ... [摘要]  真核细胞中的基因表达在转录和转录后水平受到严格调控。 mRNA转录,mRNA转录和mRNA翻译等后转录过程由RNA结合蛋白(RBPs)和非编码(nc)RNAs控制。大量的ncRNA家族包含多种调控RNA,如microRNAs和长的非编码(lnc)RNAs,但也是探索不足的一类环状RNAs。虽然三十多年前电子显微镜首次发现,但只有高通量RNA测序(RNA-seq)的出现和创新生物信息学管道的开发已经开始允许系统鉴定circRNA(Szabo和Salzman,2016;熊猫,2017b;熊猫等,2017c)。然而,通过RNA测序鉴定的真正的circRNA的验证需要其他分子生物学技术,包括常规或定量(q)聚合酶链反应(PCR)和Northern印迹分析(Jeck和Sharpless,2014)的逆转录(RT)。使用不同引物的环状RNA的RT-qPCR分析已被广泛用于检测,验证和有时定量circRNA(Abdelmohsen等人,2015和2017; Panda等人, ,2017b)。如在此详述的,设计为跨越循环RNA后接连接序列的分歧引物可以特异性扩增circRNA而不是对应的线性RNA。总之,使用不同引物的RT-PCR分析允许直接检测和定量circRNA。

【背景】CircRNAs是共价闭合的,缺少5'或3'末端的单链RNA。虽然它们的起源知之甚少,但它们可以通过称为反向剪接的过程从前体mRNA产生(Panda等人,2017d; ...

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