| Sleeping Beauty Transposon-based System for Rapid Generation of HBV-replicating Stable Cell Lines
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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 ...
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| In situ Hybridization (ISH) in Preparasitic and Parasitic Stages of the Plant-parasitic Nematode Meloidogyne spp.
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Author:
Date:
2018-03-20
[Abstract] The spatio-temporal expression pattern of a gene provides important indications to better understand its biological function. In situ hybridization (ISH) uses a labeled complementary single-stranded RNA or DNA probe to localize gene transcripts in a whole organism, a whole organ or a section of tissue. We adapted the ISH technique to the plant parasite Meloidogyne spp. (root-knot nematode) to visualize RNAs both in free-living preparasitic juveniles and in parasitic stages settled in the plant tissues. We describe each step of the probe synthesis, digoxigenin (DIG) labeling, nematode extraction from plant tissue, and ISH procedure.
[摘要] 基因的时空表达模式为更好地理解其生物学功能提供了重要的指示。 原位杂交（ISH）使用标记的互补单链RNA或DNA探针来定位整个生物体，整个器官或一部分组织中的基因转录物。 我们将ISH技术应用于植物寄生虫
【背景】到目前为止，植物寄生性线虫的稳定转化尚未成功。 ISH能够在整个装载的Meloidogyne spp中分析体内时空基因表达。线虫。这些根结线虫在土壤中以微小蚓状幼虫（J2）形式孵化并感染宿主植物根部。 J2s穿透根部并迁移到根部维管柱状细胞。幼虫定居在根部，发育成J3和J4寄生幼鱼，诱导分化专化饲养细胞。线虫最终发育成梨形雌性，将在根表面释放数百个卵。在这里，我们报告了一个详细的协议来检测准备性整体安装J2s和寄生阶段中的单个RNA分子。寄生虫阶段的ISH需要在感染根部提取线虫前一天采取额外的程序。我们描述了在线虫整个组织中使用地高辛（DIG）标记的cDNA探针检测转录物。
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| Detection and Visualization of Specific Gene Transcripts by in situ RT-PCR in Nematode-Infected Arabidopsis Root Tissue
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Author:
Date:
2015-09-20
[Abstract] This protocol describes an effective method of in situ RT-PCR that was developed to localize specific gene expression directly in thin cross-sections of nematode feeding sites induced by the cyst nematode Heterodera schachtii (H. schachtii) or the root-knot nematode Meloidogyne incognita (M. incognita) in Arabidopsis roots using DIG (Digoxigenin-11dUTP) labeling coupled with AP (alkaline phosphatase) and nitro-blue tetrazolium/5-bromo-4-chloro-3'-indolylphosphate-based detection. This method is applicable to any other Arabidopsis root tissue.
[摘要] 该方案描述了原位 RT-PCR的有效方法，其被开发用于直接在由胞囊线虫Heterodera schachtii诱导的线虫摄食位点的薄横截面中定位特异性基因表达。 在拟南芥根中（ H。schachtii ）或根结线虫 Meloidogyne incognita 使用与AP（碱性磷酸酶）和硝基蓝四唑/5-溴-4-氯-3'-吲哚磷酸盐基检测偶联的DIG（地高辛-11dUTP）标记。 该方法适用于任何其他拟南芥根组织。
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